Methods for drug discovery, disease treatment, and diagnosis using metabolomics

ABSTRACT

The small molecule profiles of cells are compared to identify small molecules which are modulated in altered states. Cellular small molecule libraries, methods of identifying tissue sources, methods for treating genetic and non-genetic diseases, and methods for predicting the efficacy of drugs are also discussed.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/421,226, filed on Oct. 25, 2002. This application is also acontinuation-in-part of U.S. patent application Ser. No. 09/835,119,entitled “Methods for Drug Discovery, Disease Treatment, and DiagnosisUsing Metabolomics,” filed on Apr. 13, 2001 now abandoned; which claimspriority to U.S. Provisional Application Ser. No. 60/239,340, entitled“Methods for Drug Discovery, Disease Treatment, and Diagnosis UsingMetabolomics” filed on Oct. 11, 2000; U.S. Provisional Application Ser.No. 60/239,541, entitled “Methods for Drug Discovery, Disease Treatment,and Diagnosis Using Metabolomics” filed on Oct. 10, 2000; U.S.Provisional Application Ser. No. 60/197,117, entitled “Small MoleculeProfiles of Cells and Methods Of Use Thereof,” filed on Apr. 14, 2000;and U.S. Provisional Application Ser. No. 60/197,085, entitled “CellularSmall Molecule Libraries,” filed on Apr. 14, 2000. The entire contentsof each of the aforementioned applications are hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Living organisms are autonomous chemical systems which include diversesets of small molecules. Small molecules found in living systemsinclude, for example, sugars, fatty acids, amino acids, nucleotides, andintermediates of metabolic and signaling pathways. Sugars are a primarysource of chemical energy for cells. The cells break the sugars downthrough a series of oxidative reactions to small sugar derivatives and,ultimately, CO₂ and H₂O. Fatty acids used for both energy storage and asmajor components of cellular membranes. Amino acids are the buildingblocks of proteins. Nucleotides are involved in intracellular signaling,energy transfer, and as the monomers of the information macromolecules,RNA and DNA.

The cellular small molecules are, generally, composed of six elements(C, H, N, O, P, S). If water is excluded, carbon compounds comprise alarge majority of the cellular small molecules. The cellular smallmolecules repeatedly use certain distinctive chemical groups, such asmethyl (CH₃), carboxyl (COOH) and amino (NH₂) groups.

Generally, most cellular small molecules are synthesized from and brokendown to the same basic compounds. Synthesis and metabolism occursthrough sequences of controlled chemical reactions, catalyzed byenzymes. Most of the metabolic reactions of the cell occur in thecytoplasm, which contains many distinctive organelles. For example, themitochondria are responsible for respiration and energy production.Mitochondria are the “power plants” of eukaryotic cells, harnessingenergy contained by combining oxygen with metabolites to make ATP. Otherorganelles of the cell include the Golgi apparatus, a system of stacked,membrane bound, flattened sacs involved in modifying, sorting andpackaging of macromolecules for secretion or for delivery to otherorganelles. The endoplasmic reticulum (ER) is a series of flattenedsheets, sacks, and tubes of membrane extending throughout the cytoplasmof eukaryotic cells. The ER membrane is in structural continuity withthe outer membrane of the nuclear envelope and specializes in thesynthesis and transport of lipids and membrane proteins.

SUMMARY OF THE INVENTION

In recent years, scientists have attempted to study cells and livingsystems through the cataloging of the entire genome of an organism(e.g., genomics). Genomics is a powerful tool, useful for identifyingand interrogating the entire inventory of genes of a living system.Recently, scientists have also attempted to identify and interrogate allthe proteins present in the cell or organism through proteomics.However, most pharmaceutical companies who study genomics and proteomicsrealize that many of their anticipated products are not proteins norgenes but small molecules.

For example, once a novel gene or target is discovered by genomics, theinvestigators must first validate the target using expensive and timeconsuming procedures which are far removed from the actual diseasestate. Examples of typical validation procedures include expressionprofiling, generating knock-out mice or transgenic mice, in situhybridization, etc. Once a target is validated, the investigatorstypically screen enormous random small molecule libraries to identifymolecules which interact with the protein targets. The identified smallmolecules typically optimized through chemical synthesis in order toobtain a marketable product.

The invention pertains, at least in part, to the generation and theanalysis of small molecule profiles of cells, cellular compartments, andspecific organelles (e.g., mitochondria, Golgi, endoplasmic reticulum,cytoplasm, nucleus, etc.) Small molecule profiles allow for theidentification and interrogation of inventories of small molecules(e.g., the metabolome) to find, for example, disease-relevant smallmolecules as well as potential targets for drug design.

Small molecule profiles of cells and organelles can be used directly toidentify drug candidates. Unlike genomics, small molecule profiling caneither eliminate entirely or accelerate the process of identifying genesand proteins associated with a disease state. In one embodiment of theinvention, the methods of the invention include, for example, comparingsmall molecule profiles of diseased cells, cellular compartments, andorganelles to standard profiles of a healthy cells, cellularcompartments, and organelles. Therefore, if a particular diseased cell,cellular compartment, or organelle was found to be deficient in aparticular compound, the deficiency may be overcome by simplyadministering the compound or an analogue thereof. Metabolomics offers anew route to the identification of potentially therapeutic agents andtargets.

Metabolomics eliminates much of the guess work surrounding genomics. Forexample, small molecule profiling allows one to investigate the verybiochemical pathway (e.g., cellular metabolites) involved in the diseasestate by comparing small molecule profiles of cells, cellularcompartments, or organelles with those of cells, cellular compartments,or organelles treated with toxins, chemical agents or other therapeuticagent (or derived from an organism treated with the agent or drug).

The invention also includes methods for identifying potential cell drugtargets (e.g., cellular components which interact with the labeled smallmolecules). This method is particularly useful because it can identifycomponents which are known to interact with disease relevant smallmolecules. Therefore, targets identified through this method are“pre-validated,” and some of the guess work surrounding the choice oftarget is eliminated. In a further embodiment, this method can be usedin conjunction with conventional genomics as a further validation stepto identify targets for further research.

Unlike genomics, small molecule profiling is not limited to diseasestates with a genetic component. Many disease states are not geneticallydetermined and genomics offers little to those suffering or at risk ofsuffering from non-genetic linked disease states. Therefore, there is aneed for a comprehensive method to study the effects of nongeneticfactors on cells and living systems.

Small molecule profiling of cells, organelles, or extracellular materialcan be used to study both genetic and non-genetically linked diseasestates. For example, methods of the invention can be used to identifyingsmall molecules associated with, for example, body weight disorders,central nervous system disorders, cardiovascular disorders,immunological disorders, oncological disorders, etc.

In addition, metabolomics can be used in tandem with genomics and/orproteomics. For example, small molecule profiles can be used to identifysmall molecules regulated, modulated, or associated with geneticmodification or alterations of cells, both engineered and naturallyoccurring.

In addition, metabolomics can also be applied to the field of predictivemedicine. For example, the invention pertains to diagnostic assays,prognostic assays, pharmacometabolomics, and the monitoring clinicaltrails which are used for prognostic (predictive) purposes to treat anindividual prophylactically, based on an individual's “metaboprint.”Unlike pharmacogenetics, which is limited to genetic factors,pharmacometabolomics is able to predict an individual's response to adrug based not only on genetic factors, but also non-genetic factors,such as other drugs in the patient's body, the patient's current stateof health, etc. Pharmacometabolomics allows for the use of a subject'ssmall molecule profile (or “metaboprint”) to deliver the right drug tothe right patient. Subjects respond differently to drugs based on theirsmall molecule profiles.

The small molecule profiling of cells, cellular compartments, particularorganelles, and/or extracellular material of the present invention canalso be used to identify individuals from minute biological samples. Themethod includes taking one or more samples from a subject anddetermining the small molecule profiles of the samples; taking a samplefrom a unknown source and determining its small molecule profile; andcomparing the two small molecule profiles to determine whether the smallmolecule profiles are from the same individual.

The invention also pertains, for example, to pharmaceutical compositionscomprised of compounds identified by the methods of the invention and apharmaceutical carrier.

In another embodiment of the invention, the invention includes a methodfor the identification of insecticides, herbicides, and othercompositions for agricultural use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows markers in subjects with amylotropiclateral sclerosis (ALS) who are not taking Riluzole.

FIG. 2 is a graph which shows markers in subjects with amylotropiclateral sclerosis (ALS) who are taking Riluzole.

FIG. 3 is a three dimensional graph showing the statistical separationof subjects. The population, ALS1, are suffering from ALS and areundergoing Riluzole treatment, while the population ALS2 are sufferingfrom ALS and not undergoing Riluzole treatment.

FIG. 4 is a partial least squares-discriminant analysis (PLS-DA) whichseparates ALS patients from their control counterparts.

FIG. 5 is shows three chromatograms generated using the HPLC-CEAS forsubjects suffering from ALS.

FIGS. 6A, 6B, and 6C are based on an HPLC analysis of the metabolome. InFIG. 6A, 39 compounds are reduced. In FIG. 6B, it is shown that 17compounds are increased in patients on Riluzole (RZ). FIG. 6B also showsthat 17 other compounds are increased in ALS patients with prominentlower motor neuron signs and slow course. In FIG. 6C, partial leastsquares-discriminant analysis was used to differentiate controls,subjects suffering from ALS on Riluzole, subjects suffering from ALS noton Riluzole and subjects suffering from lower motor neuron signs andslow course.

DETAILED DESCRIPTION OF THE INVENTION

1. Small Molecule Profiles of Cells, Cellular Compartments, andOrganelles

The invention pertains, at least in part, to the generation of smallmolecule profiles of samples, cells, and cellular compartments. Smallmolecule profiles “fingerprint” the cell or cellular compartment andidentify the presence, absence or relative quantity of small molecules.The small molecule profiles of the cells or cellular compartments may beobtained through, for example, a single technique or a combination oftechniques for separating and/or identifying small molecules known inthe art. Examples of separation and analytical techniques which can beused to separate and identify the compounds of the small moleculeprofiles include: HPLC, TLC, electrochemical analysis, massspectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), Light Scattering analysis (LS) and other methodsknown in the art. Preferably, the methods of the invention detect bothelectrically neutral as well as electrochemically active compounds.Detection and analytical techniques can be arranged in parallel tooptimize the number of molecules identified.

The term “sample” include cellular extracts from which a small moleculeprofile of the extract can be obtained. In one embodiment, the samplesare substantially free of macromolecules (e.g., large proteins andpolynucleotides with molecular weights of greater than 10,000). Thesample may be obtained from the entire cell or from specific cellularcompartments. Examples of specific cellular compartments include thecytoplasm, the mitochondria, the Golgi apparatus, the endoplasmicreticulum, the nucleus, the chloroplasts, the cytosol, etc. The term“samples” includes both isolated small molecules and mixtures of smallmolecules.

The term “cells” includes prokaryotic cells, eukaryotic cells, yeastcells, bacterial cells, plant cells, animal cells, such as, reptiliancells, bird cells, fish cells, mammalian cells. Preferred cells includethose derived from humans, dogs, cats, horses, cattle, sheep, pigs,llamas, gerbils, squirrels, goats, bears, chimpanzees, mice, rats,rabbits, etc. The term cells includes transgenic cells from cultures orfrom transgenic organisms. The cells may be from a specific tissue, bodyfluid, organ (e.g., brain tissue, nervous tissue, muscle tissue, retinatissue, kidney tissue, liver tissue, etc.), or any derivative fractionthereof. The term includes healthy cells, transgenic cells, cellsaffected by internal or exterior stimuli, cells suffering from a diseasestate or a disorder, cells undergoing transition (e.g., mitosis,meiosis, apoptosis, etc.), etc.

In a further embodiment, the samples are obtained from a specificcellular compartment. The term “cellular compartment” includesorganelles (such as mitochondria, Golgi apparatus, centrioles,chloroplasts), the nucleus, the cytoplasm (optionally including theorganelles), and other cellular regions capable of being isolated. Inone embodiment, the cellular compartment is the entire cell.

The analysis of a particular cellular compartment has many advantagesover analysis of whole cells, whole cell lysates, body fluids, etc. Forexample, often the mechanism of action of a drug, a toxic compound, etc.is directed to a specific cellular function, such as, for example, theelectron transport chain in the mitochondria, nucleic acid replicationin the nucleus, etc. By isolating the specific cellular compartment ororganelle (e.g., mitochondria, nuclei, Golgi apparatus, endoplasmicreticulum, ribosomes, etc.), it is possible to narrow the focus of theprofile to small molecules involved in the relevant pathway. Previously,metabolome studies have been complicated by the large number of chemicalspecies present in a given sample. By narrowing the scope of the studyto the particular organelle, researchers will be able to study thepathway of interest in more detail without irrelevant molecules presentin interstitial fluid, blood, spinal fluid, saliva, etc.

The term “small molecules” includes organic and inorganic moleculeswhich are present in the cell, cellular compartment, or organelle. Theterm does not include large macromolecules, such as large proteins(e.g., proteins with molecular weights over 2,000, 3,000, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, or 10,000), large nucleic acids (e.g.,nucleic acids with molecular weights of over 2,000, 3,000, 4,000, 5,000,6,000, 7,000, 8,000, 9,000, or 10,000), or large polysaccharides (e.g.,polysaccharides with a molecular weights of over 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, or 10,000). The small molecules ofthe cell are generally found free in solution in the cytoplasm or inother organelles, such as the mitochondria, where they form a pool ofintermediates which can be metabolized further or used to generate largemolecules, called macromolecules. The term “small molecules” includessignaling molecules and intermediates in the chemical reactions thattransform energy derived from food into usable forms. Examples of smallmolecules include sugars, fatty acids, amino acids, nucleotides,intermediates formed during cellular processes, and other smallmolecules found within the cell. In one embodiment, the small moleculesof the invention are isolated.

The term “metabolome” includes all of the small molecules present in agiven organism. The metabolome includes both metabolites as well asproducts of catabolism. In one embodiment, the invention pertains to asmall molecule profile of the entire metabolome of a species. In anotherembodiment, the invention pertains to a computer database (as describedbelow) of the entire metabolome of a species, e.g., an animal, e.g., amammal, e.g., a mouse, rat, rabbit, pig, cow, horse, dog, cat, bear,monkey, and, preferably, a human. In another embodiment, the inventionpertains to a small molecule library of the entire metabolome of anorganism (as described below), e.g., a mammal, e.g., a mouse, rat,rabbit, pig, cow, horse, dog, cat, bear, monkey, and, preferably, ahuman.

The language “small molecule profile” includes the inventory of smallmolecules in tangible form within a targeted cell, tissue, organ,organism, or any derivative fraction thereof, e.g., cellularcompartment, that is necessary and/or sufficient to provide informationto a user for its intended use within the methods described herein. Theinventory would include the quantity and/or type of small moleculespresent. The ordinarily skilled artisan would know that the informationwhich is necessary and/or sufficient will vary depending on the intendeduse of the “small molecule profile.” For example, the “small moleculeprofile,” can be determined using a single technique for an intended usebut may require the use of several different techniques for anotherintended use depending on such factors as the disease state involved,the types of small molecules present in a particular targeted cellularcompartment, the cellular compartment being assayed per se., etc.

The relevant information in a “small molecule profile” also may varydepending on the intended use of the compiled information, e.g. spectra.For example for some intended uses, the amounts of a particular smallmolecule or a particular class of small molecules may be relevant, butfor other uses the distribution of types of small molecules may berelevant.

The ordinarily skilled artisan would be able to determine theappropriate “small molecule profiles” for each method described hereinby comparing small molecule profiles from diseased and/or test subjectswith standard and/or healthy subjects. These comparisons can be made byindividuals, e.g., visually, or can be made using software designed tomake such comparisons, e.g., a software program may provide a secondaryoutput which provides useful information to a user. For example, asoftware program can be used to confirm a profile or can be used toprovide a readout when a comparison between profiles is not possiblewith a “naked eye”. The selection of an appropriate software program,e.g., a pattern recognition software program, is within the ordinaryskill of the art. An example of such a program is Pirouette. It shouldbe noted that the comparison of the profiles can be done bothquantitatively and qualitatively.

The small molecule profiles can be obtained from an organism sufferingfrom a disease state, genetic alteration, or any of the models discussedin more detail below. In one embodiment, the small molecule profile ofan organism is determined by using HPLC (Kristal, et al. Anal. Biochem.263:18-25 (1998)), thin layer chromatography (TLC), or electrochemicalseparation techniques (see, WO 99/27361, WO 92/13273, U.S. Pat. Nos.5,290,420, 5,284,567, 5,104,639, 4,863,873, and RE32,920). Othertechniques for determining the presence of small molecules ordetermining the identity of small molecules of the cell are alsoincluded, such as refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), Light Scattering analysis (LS) and other methodsknown in the art. The small molecule profiles of the invention may alsobe referred to as “metaboprints.” The exact combination of techniquesused to determine the small molecule profiles can be determined by

In one embodiment, the invention pertains to small molecule profilesgenerated by several methods, e.g., HPLC, TLC, electrochemical analysis,mass spectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), Light Scattering analysis (LS) and other methodsknown in the art.

The methods of the invention have several advantages over methods whichrely only on a single mode of analysis, such as electrochemicalseparation. While electrochemical separation works only for“electrochemically” active compounds, it does not effectively separateneutral molecules. The invention here relates to the use in tandem andin parallel of a multitude of these detectors. This will result in theidentification of a more comprehensive database. The detectors areusually attached to the HPLC columns where they can detect and emit aresponse due to the eluting sample and subsequently signal a peak on achromatogram. The bandwidth and height of the peaks may usually beadjusted using the coarse and fine tuning controls and the detection andsensitivity parameters may also be controlled. There are many detectorsthat can be used with the HPLC. Some detectors which can be used in themethods of the invention include: Refractive Index (RI), Ultra-Violet(UV), Fluorescent, Radiochemical, Electrochemical, Near-InfraRed(Near-IR), Mass Spectroscopy (MS), Nuclear Magnetic Resonance (NMR),Light Scattering (LS) among others.

The methods of the invention can be used to detect bothelectrochemically active molecules as well as electrochemically neutralmolecules. In a further embodiment, the invention pertains to methodswhich detect about 50% or more, about 60% or more, about 70% or more,about 75% or more, about 77.5% or more, about 80% or more, about 82.5%or more, about 85% or more, about 86% or more, about 87% or more, about88% or more, about 89% or more, about 90% or more, about 91% or more,about 92% or more, about 93% or more, about 94% or more, about 95% ormore, about 96% or more, about 97% or more, about 98% or more, about 99%or more of the small molecules of a cellular compartment (e.g.,mitochondria, chloroplast, endoplasmic reticulum, nuclei, Golgiapparatus, cytosol, etc.).

In one embodiment, HPLC columns equipped with coulometric arraytechnology can be used to analyze the samples, separate the compounds,and/or create a small molecule profiles of the samples. Such HPLCcolumns have been used extensively in the past for serum, urine andtissue analysis and are suitable for small molecule analysis (Acworth etal., 300; Beal et al., J Neurochem. 55, 1327-1339, 1990; Matson et al.,Life Sci. 41, 905-908, 1987; Matson et al., Basic, Clinical andTherapeutic Aspects of Alzheimer's and Parkinson's Diseases, vol II, pp.513-516, Plenum, N.Y. 1990; LeWitt et al., Neurology 42, 2111-2117,1992; Milbury et al., J. Wildlife Manag., 1998; Ogawa et al., Neurology42, 1702-1706, 1992; Beal et al., J. Neurol. Sci 108, 80-87, 1992,Matson et al., Clin. Chem. 30, 1477-1488, 1984; Milbury et al.,Coulometric Electrode Array Detectorsfor HPLC, pp. 125-141, VSPInternational Science Publication; Acworth et al., Am. Lab 28, 33-38,1996). HPLC columns equipped with coulometric arrays have been used forthe simultaneous analysis of the majority of low-molecule weight,redox-active compounds in mitochondria. (Anal. Biochem. 263, 18-25,1998).

For the detection and characterization of the small molecules in aneffort to create a comprehensive small molecule profiles, a multitude ofdetection methods can be used. These methods are described in moredetail below.

A. Mass Spectroscopy (MS) Detectors:

The sample compound or molecule is ionized, it is passed through a massanalyzer, and the ion current is detected. There various methods forionization. Examples of these methods of ionization include electronimpact (EI) where an electric current or beam created under highelectric potential is used to ionize the sample migrating off thecolumn, chemical ionization utilizes ionized gas to remove electronsfrom the compounds eluting from the column; and fast atom bombardmentwhere Xenon atoms are propelled at high speed in order to ionize theeluents from the column.

B. Pyrolysis Mass Spectrometry:

Pyrolysis is the thermal degradation of complex material in an inertatmosphere or vacuum. It causes molecules to cleave at their weakestpoints to produce smaller, volatile fragments called pyrolysate (Irwin1982). Curie-point pyrolysis is a particularly reproducible andstraightforward version of the technique, in which the sample, driedonto an appropriate metal is rapidly heated to the Curie-point of themetal. A mass spectrometer can then be used to separate the componentsof the pyrolysate on the basis of their mass-to-charge ratio to producea pyrolysis mass spectrum (Meuzelaar et al 1982) which can then be usedas a “chemical profile” or fingerprint of the complex material analyzed.The combined technique is known as pyrolysis mass spectrometry (PyMS).

C. Nuclear Magnetic Resonance (NMR) Detectors:

Certain nuclei with odd-numbered masses, including H and ¹³C, spin aboutan axis in a random fashion. When they are placed between poles of astrong magnet, the spins are aligned either parallel or anti-parallel tothe magnetic field, with parallel orientation favored since it isslightly lower energy. The nuclei are then irradiated withelectromagnetic radiation which is absorbed and places the parallelnuclei into a higher energy state where they become in resonance withradiation. Different spectra will be produced depending on the locationof the H or ¹³C and on adjacent molecules or elements in the compoundbecause all nuclei in molecules are surrounded by electron clouds whichchange the encompassing magnetic field and thereby alter the absorptionfrequency.

D. Refractive Index (RI):

In this method, detectors measure the ability of samples to bend orrefract light. This property for each compound is called refractiveindex. For most RI detectors, light proceeds through a bi-modular flowto a photodetector. One channel of the flow-cell directs the mobilephase passing through the column while the other directs only the otherdirects only the mobile phase. Detection occurs when the light is bentdue to samples eluting from the column, and is read as a disparitybetween the two channels. Laser based RI detectors have also becomeavailable.

E. Ultra-Violet (UV) Detectors:

In this method, detectors measure the ability of a sample to absorblight. This could be accomplished at a fixed wavelength usually 254 nm,or at variable wavelengths where one wavelength is measured at a timeand a wide range is covered, alternatively Diode Array are capable ofmeasuring a spectrum of wavelengths simultaneously. Sensitivity is inthe 10⁻⁸ to 10⁻⁹ gm/ml range. Laser based absorbance or FourierTransform methods have also been developed.

F. Fluorescent Detectors:

This method measure the ability of a compound to absorb then re-emitlight at given wavelengths. Each compound has a characteristicfluorescence. Each compound has a characteristic fluorescence. Theexcitation source passes through the flow-cell to a photodetector whilea monochromator measures the emission wavelengths. Sensitivity is in the10⁻⁹ to 10⁻¹¹ gm/ml. Laser based fluorescence detectors are alsoavailable.

G. Radiochemical Detection:

This method involves the use of radiolabeled material, for example,tritium (³H) or carbon 14 (¹⁴C). It operates by detection offluorescence associated with beta-particle ionization, and it is mostpopular in metabolite research. The detector types include homogeneousmethod where addition of scintillation fluid to column effluent causesfluorescence, or heterogeneous detection where lithium silicate andfluorescence by caused by beta-particle emission interact with thedetector cell. Sensitivity is 10⁻⁹ to 10⁻¹⁰ gm/ml.

H. Electrochemical Detection:

Detectors measure compounds that undergo oxidation or reductionreactions. Usually accomplished by measuring gains or loss of electronsfrom migration samples as they pass between electrodes at a givendifference in electrical potential. Sensitivity of 10⁻¹² to 10⁻¹³gms/ml.

I. Light Scattering (LS) Detectors:

This method involves a source which emits a parallel beam of light. Thebeam of light strikes particles in solution, and some light is thenreflected, absorbed, transmitted, or scattered. Two forms of LSdetection may be used to measure transmission and scattering.

Nephelometry, defined as the measurement of light scattered by aparticular solution. This method enables the detection of the portion oflight scattered at a multitude of angles. The sensitivity depends on theabsence of background light or scatter since the detection occurs at ablack or null background. Turbidimetry, defined as the measure of thereduction of light transmitted due to particles in solution. It measuresthe light scatter as a decrease in the light that is transmitted throughparticulate solution. Therefore, it quantifies the residual lighttransmitted. Sensitivity of this method depends on the sensitivity ofthe machine employed, which can range from a simple spectrophotometer toa sophisticated discrete analyzer. Thus, the measurement of a decreasein transmitted light from a large signal of transmitted light is limitedto the photometric accuracy and limitations of the instrument employed.

Near Infrared scattering detectors operate by scanning compounds in aspectrum from 700-1100 nm. Stretching and bending vibrations ofparticular chemical bonds in each molecule are detected at certainwavelengths. This is a fast growing method which offers severaladvantages; speed, simplicity of preparation of sample, multipleanalyses from single spectrum and nonconsumption of the sample (McClure,1994).

J. Fourier Transform Infrared Spectroscopy (FT-IR):

This method measures dominantly vibrations of functional groups andhighly polar bonds. The generated fingerprints are made up of thevibrational features of all the sample components (Griffiths 1986).FT-IR spectrometers record the interaction of IR radiation withexperimental samples, measuring the frequencies at which the sampleabsorbs the radiation and the intensities of the absorptions.Determining these frequencies allows identification of the sampleschemical makeup, since chemical functional groups are known to absorblight at specific frequencies. Both quantitative and qualitativeanalysis are possible using the FT-IR detection method.

K. Dispersive Raman Spectroscopy:

Dispersive Raman Spectroscopy is a vibrational signature of a moleculeor complex system. The origin of dispersive raman spectroscopy lies inthe inelastic collisions between the molecules composing say the liquidand photons, which are the particles of light composing a light beam.The collision between the molecules and the photons leads to an exchangeof energy with consequent change in energy and hence wavelength of thephoton.

To create a small molecule profile (or “Metaboprint”) organs, cells,cellular compartments, or organelles are homogenized in standard waysknow for those skilled in the art. Different fractionation procedurescan be used to enrich the fractions for small molecules. The smallmolecules obtained will then be passed over several fractionationcolumns. The fractionation columns will employ a variety of detectorsused in tandem or parallel to generate the small molecule profile forthe organ, cell, cellular compartment, or organelle.

For example, to generate a small molecule profile of water solublemolecules, the cell, cellular compartment, or organelle extracts will befractionated on HPLC columns with a water soluble array. The watersoluble small molecules can then be detected using fluorescence or UVdetectors to generate the small molecule profiles. Alternatively,electrochemical detectors can be used with diads to pick up redox activecompounds and the absorbance of active compounds. For generatingdetecting non water soluble molecules, hydrophobic columns can also beused to generate small molecule profiles. In addition, gaschromatography combined with mass spectroscopy, liquid chromatographycombined with mass spectroscopy, MALDI combined with mass spectroscopy,ion spray spectroscopy combined with mass spectroscopy, capillaryelectrophoresis, NMR and IR detection are among the many othercombinations of separation and detection tools which can be used togenerate small molecule profiles.

These small molecule profiles (or “metaboprints”) will be able to defineand characterize organs, cells, cellular compartments, and organelles bytheir small molecule content in both health and disease states. Theinformation generated by the small molecule profiles will be bothqualitative and quantitative.

2. Methods of Identification of Disease-Relevant Small Molecules

In another embodiment, the invention includes a method of identifyingdisease-relevant small molecules. The method includes comparing smallmolecule profiles of diseased cells, cellular compartments, ororganelles to a standard profile of a healthy cell, cellularcompartment, or organelle. The method also involves identifying thesmall molecules which are present in aberrant amounts in the diseasedsmall molecule profile. The small molecules present in aberrant amountsin the diseased cells are “disease-relevant small molecules.”

The language “disease-relevant small molecules” includes both smallmolecules present in aberrant amount in diseased small molecule profilesand, in addition, small molecules which are potentially involved indisease initiation, progression or prediction. The term also includessmall molecules which are identified using the assays for particulardiseases given below, as well as, compounds which are identified asbeing associated with particular genes of interest, also given below.The term also may include small molecules which when modulated, resultin the lessening or curing of at least one symptom of a disease. Thedisease relevant small molecules are ideal drug candidates in thescreening assays discussed elsewhere in the application.

For example, identified disease relevant small molecules may be screenedusing in vitro or in vivo assays known in the art to determinebiological activity. The biological activity of disease relevant smallmolecules can also be pinpointed by using screening assays againstprotein targets which have been implicated in the disease state. Inanother embodiment, the biological activity of disease relevant smallmolecules can be determined using cell-based assays, e.g., tumor cellassays (Lillie et al. Cancer Res. 53(13):3172-8 (1993)). The diseaserelevant small molecules can also be tested for neuronal protectionactivity by exposing primary or cultured neurons to the compounds andtoxic agents, such as glutamate, and identifying the compounds whichprotect the neurons from death. Animal models can also be used tofurther identify the biological activity of disease relevant smallmolecules. For example, animal models of Huntington's Disease,Parkinson's disease, and ALS can be used to identify small moleculesuseful as neuroprotective agents. (Kilvenyi, Nature Med. 5:347-350(1999); Mathews et al, Experimental Neurology 157:142-149 (1999)). In afurther embodiment, the disease relevant small molecules can bechemically modified to further enhance their pharmaceutical ornutriceutical properties.

The term “disease” or “disease state” includes all disease which resultor could potentially cause a change of the small molecule profile of acell, cellular compartment, or organelle in an organism afflicted withsaid disease. Examples of diseases include metabolic diseases (e.g.,obesity, cachexia, diabetes, anorexia, etc.), cardiovascular diseases(e.g., atherosclerosis, ischemia/reperfusion, hypertension, restenosis,arterial inflammation, etc.), immunological disorders (e.g., chronicinflammatory diseases and disorders, such as Crohn's disease, reactivearthritis, including Lyme disease, insulin-dependent diabetes,organ-specific autoimmunity, including multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease, contact dermatitis, psoriasis, graftrejection, graft versus host disease, sarcoidosis, atopic conditions,such as asthma and allergy, including allergic rhinitis,gastrointestinal allergies, including food allergies, eosinophilia,conjunctivitis, glomerular nephritis, certain pathogen susceptibilitiessuch as helminthic (e.g., leishmaniasis) and certain viral infections,including HIV, and bacterial infections, including tuberculosis andlepromatous leprosy, etc.), nervous system disorders (e.g.,neuropathies, Alzheimer disease, Parkinson's disease, Huntington'sdisease, amyotropic lateral sclerosis, motor neuron disease, traumaticnerve injury, multiple sclerosis, acute disseminated encephalomyelitis,acute necrotizing hemorrhagic leukoencephalitis, dysmyelination disease,mitochondrial disease, migrainous disorder, bacterial infection, fungalinfection, stroke, aging, dementia, peripheral nervous system diseasesand mental disorders such as depression and schizophrenia, etc.),oncological disorders (e.g., leukemia, brain cancer, pancreatic cancer,prostate cancer, liver cancer, stomach cancer, colon cancer, throatcancer, breast cancer, ovarian cancer, skin cancer, melanoma, etc.). Theterm also include disorders which result from oxidative stress.

The language “aberrant levels” includes any level, amount, orconcentration of a small molecule in a cell, cellular compartment, ororganelle which is different from the level of the small molecule of astandard sample.

The term “standard profile” includes profiles derived from healthycells, advantageously from a similar origin as the source. In oneembodiment, the standard profile is an average of many samples of acertain cell type and/or a certain cellular compartment. In anotherembodiment, the standard profile may be derived from a patient prior tothe onset of the disease state or from cells not affected by the diseasestate. Or, in another embodiment the standard profile can be an averageof the profiles obtained from numerous sources, e.g., the standardprofile may be an average of small molecule profiles obtained from 2 ormore subjects. The standard profile can be a small molecule profile of acertain cellular compartment or from a certain subset of cells. In oneembodiment, the invention pertains to the standard profile of healthycells. Advantageously, the small molecules with aberrant levels in thesample are identified, e.g., HPLC, TLC, electrochemical analysis, massspectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), Light Scattering analysis (LS) and other methodsknown in the art. In one embodiment, the small molecule profile of thesample, cell, or cellular compartment, is compared to the standardprofile by using subtracting one profile from the other. The compoundswhich are present in aberrant amounts can then be used in drug design toidentify deregulated cellular components. Standard profiles can also bemade of the effects of certain agents (e.g., drugs, therapeutic agents,toxins, etc.) on both healthy and diseased cells (e.g., cells diseasedwith the type of disease treated by the therapeutic agent).

Furthermore the language “standard profile” includes informationregarding the small molecules of the profile that is necessary and/orsufficient to provide information to a user for its intended use withinthe methods described herein. The standard profile would include thequantity and/or type of small molecules present. The ordinarily skilledartisan would know that the information which is necessary and/orsufficient will vary depending on the intended use of the “standardprofile.” For example, the “standard profile,” can be determined using asingle technique for an intended use but may require the use of severaldifferent techniques for another intended use depending on such factorsas the types of small molecules present in a particular targetedcellular compartment, the cellular compartment being assayed per se.,etc.

The relevant information in a “standard profile” also may vary dependingon the intended use of the compiled information, e.g. spectra. Forexample for some intended uses, the amounts of a particular smallmolecule or a particular class of small molecules of the standardprofile may be relevant, but for other uses the distribution of types ofsmall molecules small molecules of the standard profile may be relevant.

Furthermore, comparison of the standard profiles to profiles fromdiseased cells can be used to identify small molecules deregulated inthe disease state. The small molecules identified can be used to guidethe drug discovery effort. For example, the small molecules present inaberrant levels in the sample cells, can be identified and used aspharmaceutical or nutricutical agents. For example, if a patient issuffering from a disease state associated with a aberrantly low level ofa certain compound, the compound or a precursor thereof may be tested inan assay that mimics the disease state. In another embodiment, the smallmolecules present in aberrant amounts may be used as targets for drugdesign to develop agents with enhanced activity, e.g., enhanced activityto treat the disease state associated with the aberrant levels of thesmall molecule. Additionally libraries of small molecules based on thestructures of the small molecules present in aberrant amounts can beused to develop more potent therapeutics. The cellular targets andpathways could also be used to guide drug design.

In a further embodiment, the invention pertains to a method for treatinga patient with a deficiency in certain disease relevant small molecules.The method includes obtaining cells from the patient, obtaining thesmall molecule profile of either a particular organelle (e.g.,mitochondria, nucleus, cytoplasm, Golgi apparatus, endoplasmicreticulum, etc.) or a cell, comparing the small molecule profile with astandard profile, determining a deficiency in the patient's smallmolecule profile of a certain disease relevant small molecule, andadministering the disease relevant small molecule to the patient.

In a further embodiment, the invention features diagnostic assays forthe detection of disease states. For example, the method includesidentifying a small molecule which is present in aberrant amounts in aparticular disease state, e.g., by comparing standard profiles ofdiseased cells or cellular compartments with healthy cells or cellularcompartments to identify compounds which are present in aberrant amountsin the diseased cell or cellular compartment. The method also involvesdesigning a reagent that specifically reacts with the compound presentin aberrant amounts to indicate the presence or absence of the compound,and therefore, the presence or the absence of the disease. The inventionalso pertains to kits which include the reagent and instructions for itsuse to diagnose the disease.

3. Methods of Identifying the Effect of Chemical Agents on SmallMolecule Profiles of Cells, Cellular Compartments, Organelles, andExtracellular Material

In another aspect, the invention pertains to the comparison of smallmolecule profiles of cells, cellular compartments, organelles, orextracellular material with those of cells, cellular compartments,organelles, or extracellular material treated with toxins, chemicalagents or therapeutic agent (or derived from an organism treated withthe agent or drug). In one embodiment, the cells, cellular compartments,organelles, or extracellular material are diseased (or derived from adiseased organism) and are treated with a therapeutic agent which isknown to modify or treat that disease. For example, the small moleculeprofile of a cell treated with a therapeutic agent, chemical agent, ortoxin, can be compared the small molecule profile of a normal cell,e.g., a healthy cell of similar lineage, or a diseased cell of similarlineage which was not treated with the therapeutic agent, chemicalagent, or toxin. Examples of toxins include bacterial toxins such asendotoxins and exotoxins, such as cholera toxin, diptheria toxin,verotoxin, enterotoxin, etc. In a further embodiment, the cells aregenetically altered.

Extracellular material include blood, sera, spinal fluid, brain fluid,saliva, urine, semen, mucosal excretions, etc. Small molecule profilesof these extracellular materials of a particular organism may beobtained in a similar fashion to small molecule profiles of cells,cellular compartments and organelles.

In addition, subtraction profiles can be obtained by subtracting thenon-treated profile or a standard profile with the small moleculeprofile from a treated cell, cellular compartment, organelle, orextracellular fluid. The subtraction profiles can then be used toidentify certain small molecules the presence or the absence of whichmay indicate the efficacy or the toxicity of the compound. Thesubtraction profiles can be made using, for example, computer programsknown to those of skill in the art, e.g., pattern recognition softwareprogram. An example of such a program is Pirouette. It should be notedthat the comparison of the profiles can be done both quantitatively andqualitatively.

In a further embodiment, the invention pertains to certain smallmolecules which indicate the efficacy or the toxicity of the compound.The invention also applies to assays which can be developed to indicatethe presence or absence of these certain small molecules. For example,if the presence of a certain small molecule is essential for theefficacy of a particular therapeutic compound, then an assay can bedeveloped to quickly determine the presence or absence of this certainsmall molecule in cell samples treated with test compounds. This can beboth an effective and inexpensive method to determine the potentialefficacy of compounds. It can be used alone or in combination withtraditional drug screening assays such as, for example, binding assaysand other enzymatic assays.

For example, in search of molecules with anti-tumor activity, smallmolecule profiles could be taken of cells at certain intervals afterbeing treated with a known anti-tumor drug (e.g., taxol, cis platin,adriamycin, etc.). Comparison of the small molecule profiles of thesecells could lead to the identification of small molecules regulated bythese drug. The identified small molecules could then be used to guidedrug discovery by pointing to pathways which could be targeted for drugdesign or by using them as therapeutic or nutriceutical agents.Furthermore, both the targets and the identified small molecules can beused in assays of the invention described in detail in later sections.

The invention also includes a method for determining the toxicity of atest compound, e.g., a compound in development as a therapeutic agent.The method includes culturing cells, contacting a portion of the cellswith the test compound, taking small molecule profiles of both the cellscontacted with the test compound, taking the small molecule profiles ofcells not contacted with the test compound, and comparing the profilesto either each other or profiles from cells contacted with a knowntherapeutic agent or cells contacted with a known toxin. The method alsocan include a step of purifying a particular organelle of interest fromthe cells and obtaining the small molecule profile of the particularorganelle of interest (e.g., nuclei, mitochondria, Golgi apparatus,endoplasmic reticulum, ribosome, etc.). Extracellular material also maybe monitored in a similar fashion.

In a further embodiment, the invention pertains to a method for reducingside effects of drugs under development. For example, cells can becultured, contacted with the test compound, the small molecule profilecan be generated, and compared to the profiles of known toxins andtherapeutic agents. Changes then can be made to the structure of thetest compound to reduce the side effects. For example, in order to testfor liver toxicity, the compound may be incubated the in a liver cellculture to mimic the biotransformation that occurs in the liver. Thesmall molecule profiles of cells and organelles in the treated anduntreated liver cultures can be compared to the small molecule profilesof known toxins. Both the total cellular small molecule profile could becompared or the small molecule profile of a particular organelle, e.g.,mitochondria, Golgi apparatus, nuclei, ribosomes, endoplasmic reticulum,etc.

The methods of the invention are particularly useful because they offera quick and relatively inexpensive method to determine whether a certaintest compound is likely toxic to a body organ, such as the liver. Thisallows for pharmaceutical companies to quickly screen and identifycompounds which are toxic and to direct their research towards non-toxiccompounds.

The methods and small molecule profiles of the invention may also beused to rescue drugs, e.g., drugs which fail a particular step in theclinical or pre-clinical trial procedure. The failed drug can be exposedto cells or a test organism and small molecule profiles of the cells,cellular compartments, organelles, extracellular fluid, etc. can betaken and compared to those of known toxins, known therapeutic agents,etc. to pinpoint the reason for failure of the drug. Small moleculeprofiles of various organs can also be taken if it is advantageous forthe study (e.g., small molecule profiles can be taken from muscle,brain, retinal, nerve, heart, lung, stomach, colon, skin, breast, fattytissue, blood, etc. )Then the drug can be redesigned to avoid the itsprevious adverse effects.

The methods and small molecule profiles of the invention can also beused to “reposition” drugs.

The term “reposition” refers to discovering new uses for an agent. Inone embodiment, a dose of an agent is administered to a subject (e.g., ahuman or other animal, healthy or diseased) and small molecule profilesare then taken from various organs, tissues, cells, cellularcompartments, organelles, and/or extracellular fluid of the subject todetermine what tissues, organs, cells, cellular compartments,organelles, and/or extracellular fluids are being affected by theadministration of the agent.

4. Methods of Identifying Small Molecules Associated with Body WeightDisorders

The invention also pertains to methods for identifying small moleculesassociated with, for example, body weight disorders such as obesity.Examples of methods for identifying small molecules associated with bodyweight disorders are described below. The following experiment aredirected to the identification of small molecules associated with shortterm appetite control. These experiments can be used to identify smallmolecules involved in signaling hunger and satiety.

In one embodiment, test subjects, preferably mice, will be fed normallyprior to the initiation of the experiment, and then divided into onecontrol and two experimental groups. The control group will then bemaintained on ad lib nourishment, while the first experimental group(“fasted group”) will be fasted, and the second experimental group(“fasted-refed group”) will initially be fasted, and will then beoffered a highly palatable meal shortly before the collection of tissuesamples. Each test animal will be weighed immediately prior to andimmediately after the experiment. Small molecule profiles will be takenof each mouse from each group. The profiles of each group will beaveraged and compared to those of different groups. Example 2, below,demonstrates the use of such short term appetite experiments to identifysmall molecules which are present in different amounts in control versusfasting and versus refed animals.

Other experiments which may be used for the identification of cellularsmall molecules involved in, for example, body weight disorders, areexperiments designed to analyze small molecules which may be involvedgenetic obesity. In the case of mice, for example, such experiments mayidentify small molecules regulated by the ob, db, and/or tub geneproducts. In the case of rats, for example, such paradigms may identifysmall molecules regulated by the fatty (fa) gene product.

In one embodiment of such an experiment, test subjects may includeob/ob, db/db, and/or tub/tub experimental mice and lean littermatecontrol animals. The animals would be offered normal nourishment for agiven period, after which tissue samples would be collected foranalysis. Example 2, below, demonstrates the use of such genetic obesityparadigms in identifying small molecules which are present in differentamounts in the small molecule profiles of obese versus lean animals.

In additional experiments, ob/ob, db/db, and/or tub/tub experimentalmice and lean control animals may be used as part of the short termappetite control experiments discussed above, or in other experimentsdiscussed herein, such as set-point experiments and drug relatedexperiments.

Experiments which may be used for the identification of small moleculesinvolved in body weight disorders may include experiments designed toidentify those small molecules which may be regulated in response tochanges in body weight, e.g., “set point experiments”.

In one experiment, test subjects, preferably mice, will be fed normallyprior to the initiation of the experiment, and then divided into onecontrol and two experimental groups. The control group will then bemaintained on an ad lib diet of normal nourishment in order to calculatedaily food intake. The first experimental group (“underweight group”)will then be underfed by receiving some fraction of normal food intake,60-90% of normal, for example, so as to reduce and maintain the group'sbody weight to some percentage, for example 80%, of the control group.The second experimental group (“overweight group”) will be overfed byreceiving a diet which would bring the group to some level above that ofthe control, for example 125% of the control group. Tissue samples willthen be obtained for analysis to identify small molecules which arepresent in different amounts in control versus overweight and/orunderweight conditions.

Additionally, human subjects may be used for the identification ofobesity-associated small molecules. In one embodiment of such anexperiment, tissue samples may be obtained from obese and lean humansubjects and analyzed for the presence of small molecules which arepresent in different amounts in the tissue, cells, or cellularorganelles of one group as opposed to another (e.g. differentiallypresent in lean versus obese subjects). In another embodiment, obesehuman subjects may be studied over the course of a period of weightloss, achieved through food restriction. Tissue from these previouslyobese subjects may be analyzed for differing amounts of small moleculesrelative to tissue obtained from control (lean, non-previously obese)and obese subjects.

Experiments may also be designed to identify of small molecules involvedin body weight disorders and may also may include experiments designedto identify small molecules associated with body weight disordersinduced by some physical manipulation to the test subject, such as, forexample, hypothalamic lesion-induced body weight disorders. For example,bilateral lesions in the ventromedial hypothalamus (VMH) of rodents maybe utilized to induce hyperphagia and gross obesity in test subjects,while bilateral lesions in the ventrolateral hypothalamus (VLH) ofrodents may be used to induce aphagia in test subjects. In suchexperiments, tissue from hypothalamic-lesioned test subjects and fromcontrol subjects would be analyzed for the identification of smallmolecules which are present in different amounts in control versuslesioned animals.

Drugs known to affect (e.g., ameliorate) human or animal body weightand/or appetite (such as short term appetite) may be incorporated intothe experiments designed to identify small molecules which are involvedin body weight disorders and/or body weight or appetite regulation.These compounds may include known therapeutics, as well as compoundsthat are not useful as therapeutics due to, for example, their harmfulside effects. Among the categories of control and test subjects whichmay be used in such experiments are, for example, lean subjects, obesesubjects, and obese subjects which have received the drug of interest.In variations of the experiment, subjects such as these may be fed anormal ad lib diet, a caloric restriction maintained diet, or a caloricrestriction ad lib diet. Control and test subjects may additionally bepairfed i.e., the control and test subjects may be fed via a coupledfeeding device such that both control and test subjects receiveidentical amounts and types of food).

5. Methods of Identifying Small Molecules Associated with ImmunologicalDiseases

The invention also pertains to methods for identifying small moleculesassociated with, for example, normal and abnormal immune responses.Examples of methods for identifying small molecules associated withimmune responses are described below. The following experiment aredirected to the identification of small molecules which aredifferentially present within and among TH cell subpopulations,including but not limited to TH1 and TH2 subpopulations. Such smallmolecules can be involved in, for example, TH cell subpopulationdifferentiation, maintenance, and/or effector function, and in TH cellsubpopulation-related disorders. For example, TH cells can be induced todifferentiate into either TH1 or TH2 states, can be stimulated with, forexample, a foreign antigen, and can be collected at various pointsduring the procedure for analysis of their small molecule profiles. Thisexample is merely meant to be illustrate several experiments which canbe done using small molecule profiles to determine small moleculesassociated with immunological disorders. This example is not intended tolimit the invention to the specific types of cells or subjects discussedin this section.

In one experiment, transgenic animals, preferably mice, will be usedwhich have been engineered to express a particular T cell receptor, suchthat the predominant T cell population of the immune system of such atransgenic animal recognizes only one antigen. Such a system will beused because it provides a source for a large population of identical Tcells whose naivete can be assured, and because its response to thesingle antigen it recognizes is also assured. T helper cells can beisolated from such a transgenic animal can then be induced, in vitro, todifferentiate into TH cell subpopulations such as TH1, TH2, or TH0 cellsubpopulations. In one embodiment, one T helper cell group (the TH1group) is exposed to IL-12, a cytokine known to induce differentiationinto the TH1 state, a second T helper cell group (the TH2 group) isexposed to IL-4, a cytokine known to induce differentiation into the TH2state, and a third group is allowed, by a lack of cytokine-mediatedinduction, to enter a TH-undirected state. Small molecule profiles ofeach type of cells can then be taken and compared.

In another experiment, mature TH cell clones can be used, such as TH1and TH2 and TH1-like and TH2-like cell lines, preferably human celllines. Such TH cell lines can include, but are not limited to thefollowing well known murine cell lines: Doris, AE7, D10.G4, DAX, D1.1and CDC25. Such T cell lines can be derived from normal individuals aswell as individuals exhibiting TH cell subpopulation-related disorders,such as, for example, chronic inflammatory diseases and disorders, suchas Crohn's disease, reactive arthritis, including Lyme disease,insulin-dependent diabetes, organ-specific autoimmunity, includingmultiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishrnaniasis) and certainviral infections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

The TH cell clones can be stimulated in a variety of ways. Suchstimulation methods include, but are not limited to, pharmacologicalmethods, such as exposure to phorbol esters, calcium ionophores, orlectins (e.g., Concanavalin A), by treatment with antibodies directedagainst T-cell receptor epitopes (e.g., anti-CD3 antibodies) orexposure, in the presence of an appropriate antigen presenting cell(APC), to an antigen that the particular TH cells are known torecognize. Following such primary stimulation, the cells can bemaintained in culture without stimulation and, for example, in thepresence of IL-2, utilizing standard techniques well known to those ofskill in the art. The cells can then be exposed to one or moreadditional cycles of stimulation and maintenance. The small moleculeprofiles of the cells o cellular compartments can be taken at any timeduring the process of the stimulation in this experiment.

A third experiment can also be used to discover determine smallmolecules present in different amounts. In vivo stimulation of animalmodels forms the basis for this experiment. The in vivo nature of thestimulation can prove to be especially predictive of the analogousresponses in living patients. Stimulation can be accomplished via avariety of methods. For example, animals, such as transgenic animalsdescribed earlier, can be injected with appropriate antigen andappropriate cytokine to drive the desired TH cell differentiation.Draining lymph nodes can then be harvested at various time points afterstimulation. Lymph nodes from, for example, TH1-directed animals can becompared to those of TH2-directed animals. A wide range of animalmodels, representing both models of normal immune differentiation andfunction as well as those representing immune disorders can be utilizedfor this in vivo experiment.

Cell or organelle samples can be collected during any point of such aprocedure for small molecule profiling. For example, cells or organellescan be obtained following any stimulation period and/or any maintenanceperiod. Additionally, the cells or organelles can be collected duringvarious points during the TH cell differentiation process. The smallmolecule profiles of the cells or organelles can be compared using themethods outlined in the Examples. For example, small molecule profilesfrom TH0, TH1 and TH2 groups isolated at a given time point can then beanalyzed and compared. Additionally, small molecule profiles fromstimulated and non-stimulated cells within a given TH cell group canalso be compared and analyzed. Further, small molecule profiles fromundifferentiated TH cells can be compared to small molecule profilesfrom cells at various stages during the differentiative process whichultimately yields TH cell subpopulations.

6. Methods of Identifying Small Molecules Associated with CardiovascularDisorders

The small molecule profiles of the invention can be used to identifysmall molecules which are relevant to cardiovascular disease.

According to the invention, profiles are generated for small moleculespresent in endothelial cells or endolethial cell organelles subject tofluid shear stress in vitro. Shear stress may be responsible for theprevalence of atherosclerotic lesions in areas of unusual circulatoryflow.

Cell cultures are exposed to fluid shear stress which is thought to beresponsible for the prevalence of atherosclerotic lesions in areas ofunusual circulatory flow. Unusual blood flow also plays a role in theharmful effects of ischemia/reperfusion, wherein an organ receivinginadequate blood supply is suddenly reperfused with an overabundance ofblood when the obstruction is overcome.

Cultured HUVEC monolayers are exposed to laminar shear stress byrotating the culture in a specialized apparatus containing liquidculture medium (Nagel et al., 1994, J. Clin. Invest. 94: 885-891).Static cultures grown in the same medium serve as controls. After acertain period of exposure to shear stress, experimental and controlcells will be harvested, organelles isolated and small molecule profileswill be generated to identify molecules which are present in exposedversus control cells.

In experiments designed to identify small molecules which are involvedin cardiovascular disease, compounds such as drugs known to have anameliorative effect on the disease symptoms may be incorporated into theexperimental system. Such compounds may include known therapeutics, aswell as compounds that are not useful as therapeutics due to theirharmful side effects. Test cells that are cultured, for example, may beexposed to one of these compounds and analyzed for different smallmolecule profiles with respect to untreated cells, according to themethods described below in the Examples. In principle, according to theparticular experiment, any cell type involved in the disease may betreated at any stage of the disease process by these therapeuticcompounds.

Test cells may also be compared to unrelated cells (e.g., fibroblasts)that are also treated with the compound, in order to screen out genericeffects on small molecule profiles that may not be related to thedisease. Such generic effects might be manifest by changes in smallmolecule profiles that are common to the test cells and the unrelatedcells upon treatment with the compound.

By these methods, the small molecules upon which these compounds affectcan be identified and used in the assays described below to identifynovel therapeutic compounds for the treatment of cardiovascular disease.

In another experiment, small molecules are identified from monocytesfrom human subjects. This experiment involves differential treatment ofhuman subjects through the dietary control of lipid consumption. Thehuman subjects are held on a low fat/low cholesterol diet for threeweeks, at which time blood is drawn, monocytes are isolated according tothe methods routinely practiced in the art, organelles, such asmitochondria, nuclei, and the cytosol, are isolated and profiles aregenerated. These same patients are subsequently switched to a highfat/high cholesterol diet and monocyte organelles are purified again.The patients may also be fed a third, combination diet containing highfat/low cholesterol and monocyte organelles may be purified once again.The order in which patients receive the diets may be varied. The smallmolecules of the organelles derived from patients maintained on two ofthe diets, or on all three diets, may then be compared and analyzed.

In addition to the detection of different small molecule profiles inmonocytes, paradigms focusing on endothelial cells may be used to detectsmall molecules involved in cardiovascular disease. In one experiment,human umbilical vein endothelial cells (HUVEC's) are grown in vitro.Experimental cultures will then be treated with human IL-1β, a factorknown to be involved in the inflammatory response, in order to mimic thephysiologic conditions involved in the atherosclerotic state.Alternatively experimental HUVEC cultures may be treated withlysophosphatidylcholine, a major phospholipid component of atherogeniclipoproteins or oxidized human LDL. Control cultures are grown in theabsence of these compounds. After a certain period of treatment,experimental and control cells will be harvested and small moleculeprofiles will be taken of the cells and/or organelles and analyzed.

7. Methods of Identifying of Small Molecules Associated with CentralNervous System and Other Neurological and Neurodegenerative Disorders

The small molecule profiles of the invention can be used to identifysmall molecules which are relevant to central nervous system and otherneurological and neurodegenerative disorders. Examples of such disordersinclude, for example, neuropathies, Alzheimer disease, Parkinson'sdisease, Huntington's disease, amyotropic lateral sclerosis, motorneuron disease, traumatic nerve injury, multiple sclerosis, acutedisseminated encephalomyelitis, acute necrotizing hemorrhagicleukoencephalitis, dysmyelination disease, mitochondrial disease,migrainous disorder, bacterial infection, fungal infection, stroke,aging, dementia, peripheral nervous system diseases and mental disorderssuch as depression and schizophrenia, etc.

One method for identifying small molecules which are relevant to centralnervous system and other neurological and neurodegenerative disorders,is to compare the small molecule profiles of a diseased cell, cellularcompartment or organelle of a diseased organism to a small moleculeprofile of a healthy cell, cellular compartment, or organelle (e.g., astandard small molecule profile) For example, the cells can be derivedfrom the subjects' brain, muscle, retinal, nerve tissue, spinal fluid,blood, etc.

The diseased organism can be either a human or animal patient sufferingfrom a neurological disorder or from an animal model of such a disorder.In certain embodiments, the invention pertains to the small moleculeswhich are found in aberrant amounts in the small molecule profiles ofdiseased cells. In other embodiments, the invention pertains to thesmall molecule subtraction profiles of particular neurological disorders(e.g., subtraction profiles of the diseased small molecule profilecompared to the standard small molecule profile, etc.).

8. Methods of Identifying Small Molecules Associated with OncologicalDisorders

In one embodiment, the invention pertains to methods of identifyingsmall molecules associated with oncological disorders, e.g., canceroustumors, leukemia, lymphoma, etc.

In one embodiment, small molecules associated with an oncologicaldisorder are identified by comparing small molecule profiles ofcancerous tissue with normal tissue. In a further embodiment, the tissueis from the same individual, e.g., normal and malignant prostate tissuesare excised from a mammalian subject, e.g., mouse, rat, or human. Smallmolecule profiles of cells, cellular compartments, or organelles of thenormal tissue is compared with the corresponding small molecule profilesof the malignant tissue. When the small molecule profiles are compared,certain small molecules may appear to be present in aberrant amounts inthe cancerous tissue.

The invention also pertains to methods for detecting aberrant amounts ofthe identified compound in other tissue, e.g., the methods of theinvention can be used to develop a reagent that specifically reacts withcancerous tissue.

9. Methods of Identifying Small Molecules Regulated by Genes of Interest

In another embodiment, the invention pertains to methods of identifyingsmall molecules regulated, modulated, or associated with geneticmodification or alterations of cells, both engineered and naturallyoccurring. The identified small molecules can be used, for example, todetermine the function of unknown genes in functional genomics. Forexample, the comparison of the small molecules found in geneticallyaltered cells can be used to elucidate the function of any given gene.For example, the invention pertains to a method for identifying smallmolecules associated with expression vectors of interest by comparingthe small molecules of host cells expressing an expression vector to thesmall molecules of host cells not expressing the expression vector. Inone embodiment, the expression vector comprises a portion or fragment ofthe genome, e.g., human genome. In another embodiment, the expressionvector may be known to be associated with a particular disease state.The small molecules of the cells with and with out the expression vectorexpressed can be used to identify small molecules of interest, pathwaysof interest, and targets for drug design and/or future study.

In a further embodiment, the small molecules of the cells are identifiedby through separation techniques such as HPLC, mass spectroscopy andcoulometric array technology to create small molecule profiles (see, forexample, Kristal, B. S. et al. Anal. Biochem. 263:18-25 (1998)). Theresulting small molecule profile can then be compared to the smallmolecule profile of other cells, e.g., cells not genetically modified.

The term “vector” includes nucleic acid molecules capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc.

The recombinant expression vectors of the invention can be designed forexpression in prokaryotic or, preferably, eukaryotic host cells. Forexample, the vectors can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990).

Expression of vectors in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins.Examples of inducible non-fusion E. coli expression vectors include pTrc(Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 60-89). Target gene expression from the pTrc vectorrelies on host RNA polymerase transcription from a hybrid trp-lac fusionpromoter. Target gene expression from the pET 11d vector relies ontranscription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(In Vitrogen Corp, San Diego, Calif.).

Alternatively, the vector can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of vectors in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In a preferred embodiment, a nucleic acid of the interest is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The terms “host cell” and “recombinant host cell” are usedinterchangeably. These cells include not only the particular subjectcell but to the progeny or potential progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term as used herein.

For this method, a host cell can be any prokaryotic or eukaryotic cell.For example, a protein of interest can be expressed in bacterial cellssuch as E. coli, insect cells, yeast or, preferably, mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitablehost cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. The terms“transformation” and “transfection” include a variety of art-recognizedtechniques for introducing foreign nucleic acid (e.g., DNA) into a hostcell, including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, or electroporation.Suitable methods for transforming or transfecting host cells can befound in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd,ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as the gene or a separate vector. Cells stably transfected withthe introduced nucleic acid can be identified by drug selection (e.g.,cells that have incorporated the selectable marker gene will survive,while the other cells die).

Furthermore, in yet another embodiment, the invention also pertains tomethods for identifying small molecules regulated by a gene expressed ina particular host cell. In this embodiment, the gene is removed,functionally disrupted, otherwise not expressed in the cell and thesmall molecules of the cell are compared to those of a similar cellwherein the gene is expressed. The small molecules which are regulated,modulated or associated with this gene can then be identified by thecomparison of the small molecules of the cells with and without the geneexpressed. The small molecules which are present in aberrant amounts canthen be used to identify pathways, targets, and other small moleculesassociated with this gene, using methods of the invention.

To functionally disrupt a gene of a cell, a vector is prepared whichcontains at least a portion of a gene of interest into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the gene of interest. The gene of interest can bea human gene, or a non-human homologue of a human gene. In anembodiment, the vector is designed such that, upon homologousrecombination, the endogenous gene of interest is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous gene of interest ismutated or otherwise altered but still encodes, for example, afunctional protein (e.g., the upstream regulatory region can be alteredto thereby alter the expression of the endogenous protein). In thehomologous recombination vector, the altered portion of the gene ofinterest is flanked at its 5′ and 3′ ends by additional nucleic acidsequence of the gene of interest to allow for homologous recombinationto occur between the exogenous gene of interest carried by the vectorand an endogenous gene of interest in a cell. The additional flankingnucleic acid sequence should be of sufficient length for successfulhomologous recombination with the endogenous gene. Typically, severalkilobases of flanking DNA (both at the 5′ and 3′ ends) are included inthe vector (see e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell51:503 for a description of homologous recombination vectors). Thevector is introduced into a cell line (e.g., by electroporation) andcells in which the introduced gene of interest has homologouslyrecombined with the endogenous gene of interest are selected (see e.g.,Li, E. et al. (1992) Cell 69:915). The small molecules of the cells canthen be compared to cells with out the gene of interest disrupted, thusidentifying small molecules associated with the gene of interest.

10. Assays for Identifying Potential Cell Drug Targets Using LabeledDisease Relevant Small Molecules

In another embodiment, the invention also pertains to methods foridentifying potential cell drug targets (e.g., cellular components whichinteract with the labeled small molecules). This method is particularlyuseful because it can identify components which are known to interactwith disease relevant small molecules. Therefore, targets identifiedthrough this method are “pre-validated,” and some of the guess worksurrounding the choice of target is eliminated. In a further embodiment,this method can be used in conjunction with conventional genomics as afurther validation step to identify targets for further research.

The method includes obtaining a cell from a source, obtaining samples ofsmall molecules from the cell; testing the samples for biologicalactivity; identifying the biologically active small molecules of thesamples; labeling the biologically active small molecules; contactingthe labeled small molecules with cellular components; and identifyinginteractions between cellular components and said labeled smallmolecules. The invention includes the identified cell drug targets aswell as the identified biologically active small molecules.

In another embodiment, the invention includes a method for identifyingpotential cell drug targets. The method includes contacting a labeleddisease relevant small molecule with cellular components; andidentifying interactions between said cell components and the labeleddisease-relevant small molecule.

The labeled small molecules also include labeled “disease-relevant smallmolecules,” identified by any of the techniques described herein (e.g.,comparison of small molecule profiles in healthy and diseased cells,etc.). In another embodiment, the method includes contacting a labeleddisease relevant small molecule with cellular components, andidentifying the interactions between the cellular components and thelabeled disease relevant small molecule.

The term “label” includes any moieties or molecules which enhance theability of the labeled small molecules to be detected. Examples ofsuitable labels are well known in the art. radiolabels and fluorescentlabels. The term “label” includes direct labeling of the small moleculeby radiolabeling, coupling (i.e., physically linking) a detectablesubstance (e.g., a fluorescent moiety) to the small molecule, andindirect labeling of the small molecule by reacting the small moleculewith another reagent that is directly labeled. Examples of indirectlabeling include detection of a small molecules by labeling it withbiotin such that it can be detected with fluorescently labeledstreptavidin. In one embodiment, the small molecules are fluorescentlylabeled or radiolabeled.

The term “cellular components” includes material derived from cells. Thecellular components can be purified or crude cellular extracts. Thecellular components can be derived from one type of cell, or even aspecific cellular compartment such as an organelle (e.g., mitochondria,nucleus, cytoplasm). Furthermore, the term includes both naturalproteins found within biological systems and chimeric and otherengineered proteins. In one embodiment, the term “cellular component”includes cellular receptors. The term also includes natural andunnatural polysaccharides and nucleic acids. In one embodiment, the term“cellular component” is a crude cellular extract from a human cell. Theterm “cellular component” includes “targets.”

Samples of the invention that bind to cellular components can beidentified by preparing a reaction mixture of the cellular componentsand the samples under conditions and for a time sufficient to allow thecomponents and the sample to interact and bind, thus forming a complexwhich can be removed and/or detected in the reaction mixture. Thecellular components used can vary depending upon the goal of thescreening assay. In one embodiment, the sample of the invention is anisolated, labeled small molecule, e.g., a disease relevant smallmolecule, a small molecule with biological activity or another smallmolecules which is present in aberrant levels in disease states. Theassay can be used to determine which cellular components the smallmolecule interacts with. The identified cellular components whichinteract with the small molecule can then be used for drug design.

In a further embodiment, the cellular components are a nucleic acidarray. High density arrays of nucleic acids (such as cDNA's andsynthetic oligonucleotides) allow for a high degree of automation,repetitive analysis and duplication at minimal cost (Fraser,Electrophoresis, 18:1207-1215 (1997)). The development of recenttechnology has provided methods for making very large arrays ofoligonucleotide probes in very small areas (see, for example, U.S. Pat.No. 5,143,854, WO 90/15070 and WO 92/10092, each of which isincorporated herein by reference). In one embodiment, the nucleic acidsof the array are human genes. Examples of nucleic acid arrays includethose mentioned in U.S. Pat. Nos. 6,027,880 and 5,861,242. The nucleicacids also can be representative of RNA molecules present in a cell,tissue or organ (e.g., the “transcriptome”, see Hoheisel, J. et al.Trends Biotechnol. 15:465-469 (1997); Velculescu, Cell, 88:243-251(1997)). In one embodiment, the nucleic acids are in array.

In another further embodiment, the cellular components are a proteinarray. Examples of protein arrays include those employing conventionalprotein separation techniques, such as 2-dimensional gelelectrophoresis, chromatographic procedures (e.g., FPLC, SMART byPharmacia, Uppsala, Sweden), capillary electrophoretic techniques andmass spectrometry. In another embodiment, the protein array is a soup ofproteins that contains a significant portion of the diversity encoded bya genome (see WO 99/39210).

In a further embodiment, the cellular components are a 2D protein gel.The 2D protein gel may be a complete or an incomplete set of the proteinmolecules present in a cell, tissue or organ (e.g., the proteome, seeSagliocco, et al. Yeast 12, 1519-1534 (1996); Shevalanko, et al. Porch.Nat. Acad. Sci. 93, 14440-14445 (1996)). Labeled biologically activesmall molecules previously identified through methods of the inventioncan then be contacted with the 2D gels and interactions between thelabeled small molecules and the protein of the 2D gel can be detected.

The proteins identified through this method can then be further testedfor biological activity, e.g., biological activity relating to that ofthe small molecule, e.g., through knock-out mice, inhibition studies,and other techniques known in the art. Furthermore, the identifiedproteins can then be used in drug design to identify other molecules(either naturally occurring or chemically synthesized) which bind orinteract with the protein which may have advantageous characteristics(e.g., enhanced biological activity, less toxic side effects).

11. Predictive Medicine and Pharmacometabolomics

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, pharmacometabolomics, andmonitoring clinical trails are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningsmall molecule profiles, in the context of a biological sample (e.g.,blood, serum, cells, tissue, cellular organelles) to thereby determinewhether an individual is afflicted with a disease or disorder, or is atrisk of developing a disorder, associated with aberrant levels of smallmolecules. The invention also provides for prognostic (or predictive)assays for determining whether an individual is at risk of developing adisorder associated with relevant small molecules. For example, aberrantlevels of small molecules can be profiled from a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with a relevant small molecule.

Another aspect of the invention provides methods for determining smallmolecule profiles of an individual to thereby select appropriatetherapeutic or prophylactic agents for that individual (referred toherein as “pharmacometabolomics”). Pharmacometabolomics allows for theselection of agents (e.g., drugs) for therapeutic or prophylactictreatment of an individual based on the small molecule profile of theindividual (i.e., the individual's “metaboprint”). The metaboprint ofthe individual is examined to predict what the person's reaction to aparticular therapeutic compound will be. Yet another aspect of theinvention pertains to monitoring the influence of agents (e.g., drugs orother compounds) on the small molecule profiles of the patients inclinical trials.

Pharmacometabolomics is similar to pharmacogenomics but it is also ableto taken in to account environmental and other non-genetic factors(e.g., other drugs, etc.) which may affect an individual's response to aparticular therapeutic compound. Pharmacometabolomics can be used aloneor in combination with pharmacogenomics to predict an individual'sreaction to a particular drug based upon their metaboprint (e.g., smallmolecule profile) and/or their genotype.

Pharmacometabolomics is particularly useful because it provides an earlywarning sign, due to its capability of detecting aberrant smallmolecules long before any disease symptoms or predisposed phenotypes arenoticed.

A. Diagnostic Assays

In one embodiment, the invention pertains to a method for facilitatingthe diagnosis of a disease state of a subject. The method includesobtaining a small molecule profile from a subject suspected of havingand/or having a disease state, and comparing the small molecule profilefrom the subject to a standard small molecule profile.

The invention provides a method of assessing small molecule profiles,especially aberrant small molecule profiles. Aberrant small moleculeprofiles (e.g., excessive amounts of a particular molecule, deficientamounts of a particular molecule, the presence of a small molecule notusually present, etc.) may indicate the presence of a disease state.More generally, aberrant small molecule profiles may indicate theoccurrence of a deleterious or disease-associated metaboprintcontributed by small molecules present in aberrant amounts.

The standard small molecule profile can be obtained from healthysubjects or subjects afflicted with the disease state which is thesubject is suspected of having. The small molecule profiles can be takenfrom a particular organ, tissue, or combinations or organs or tissues.The small molecule profiles can also be taken of cells, cellularcompartments, particular organelles, or extracellular material.

The term “disease state” includes any states which are capable of beingdetected metabolomically by comparing small molecule profiles of asubject having the disease to a standard small molecule profile.Examples of disease states include, but are not limited to, includemetabolic diseases (e.g., obesity, cachexia, diabetes, anorexia, etc.),cardiovascular diseases (e.g., atherosclerosis, ischemia/reperfusion,hypertension, restenosis, arterial inflammation, etc.), immunologicaldisorders (e.g., chronic inflammatory diseases and disorders, such asCrohn's disease, reactive arthritis, including Lyme disease,insulin-dependent diabetes, organ-specific autoimmunity, includingmultiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis) and certainviral infections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy, etc.), nervous system disorders(e.g., neuropathies, Alzheimer disease, Parkinson's disease,Huntington's disease, amyotropic lateral sclerosis, motor neurondisease, traumatic nerve injury, multiple sclerosis, acute disseminatedencephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis,dysmyelination disease, mitochondrial disease, migrainous disorder,bacterial infection, fungal infection, stroke, aging, dementia,peripheral nervous system diseases and mental disorders such asdepression and schizophrenia, etc.), oncological disorders (e.g.,leukemia, brain cancer, pancreatic cancer, prostate cancer, livercancer, stomach cancer, colon cancer, throat cancer, breast cancer,ovarian cancer, skin cancer, melanoma, etc.). The term also includedisorders which result from oxidative stress.

The term “subject” includes humans, animals, and plants. In oneembodiment, the subject is a human suffering from or at risk ofsuffering from a disease state.

The invention also encompasses kits for detecting the presence of aparticular relevant small molecule in a biological sample (a testsample). Such kits can be used to determine if a subject is sufferingfrom or is at increased risk of developing a disorder associated withthe relevant small molecule (e.g., drug resistance). For example, thekit can comprise a labeled compound or agent capable of detecting therelevant small molecule in a biological sample and means for determiningthe amount of the relevant small molecule in the sample (e.g., anantibody against the relevant small molecule another molecular orchemical sensor). Kits may also include instruction for observing thatthe tested subject is suffering from or is at risk of developing adisorder associated with the relevant small molecule if the amount ofthe relevant small molecule is above or below a normal level.

The kit may also comprise, e.g., a buffering agent, a preservative, or astabilizing agent. The kit may also comprise components necessary fordetecting the detectable agent (e.g., a substrate). The kit may alsocontain a control sample or a series of control samples which can beassayed and compared to the test sample contained. Each component of thekit is usually enclosed within an individual container and all of thevarious containers are within a single package along with instructionsfor observing whether the tested subject is suffering from or is at riskof developing a disorder associated with the relevant small molecule.

B. Prognostic Assays

The invention also pertains to a method for predicting whether a subjectis prediposed to having a disease state. The method includes obtaining asmall molecule profile from the subject; and comparing the smallmolecule profile from the subject to a standard small molecule profile,thereby predicting whether a subject is prediposed to having a diseasestate.

The methods described herein can furthermore be used as diagnostic orprognostic assays to identify subjects having or at risk of developing adisease or disorder associated with aberrant small molecule profiles.For example, the assays described herein, such as the precedingdiagnostic assays or the following assays, can be utilized to identify asubject having or at risk of developing a disorder associated with anaberrant small molecule profile, such as drug resistance of tumor cells.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing such a disease or disorder.Thus, the present invention provides a method in which a test sample isobtained from a subject and a small molecule profile is taken, whereinan aberrant small molecule profile is diagnostic for a subject having orat risk of developing a disease or disorder associated with an aberrantsmall molecule profile. The term “test sample” is a biological sampleobtained from a subject of interest. For example, a test sample can be abiological fluid (e.g., serum, blood, saliva, etc.), cell sample, ortissue. Advantageously, the test sample may consist of cells,extracellular material, or individual organelles, e.g., mitochondria,nuclei, Golgi apparatus, endoplasmic reticulum, ribosomes, chloroplasts,etc.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g.,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with anaberrant small molecule profile. For example, such methods can be usedto determine whether a subject can be effectively treated with aspecific agent or class of agents (e.g., agents of a type which effectthe small molecule profile in particular ways). Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated with anaberrant small molecule profile in which a test sample is obtained andan aberrant small molecule profile is detected (e.g., wherein thepresence or relative quantity of particular relevant small molecules isdiagnostic for a subject that can be administered the agent to treat adisorder associated with the aberrant small molecule profile). In someembodiments, the foregoing methods provide information useful inprognostication, staging and management of particular states that arecharacterized by altered small molecule profiles and thus by aparticular metaboprint. The information more specifically assists theclinician in designing treatment regimes to eradicate such particularstates from the body of an afflicted subject.

The methods of the invention can also be used to detect the presence orabsence of relevant small molecules, thereby determining if a subject isat risk for a disorder associated with this relevant small molecule. Forexample, the presence or absence of relevant small molecules, mayindicate whether the process of developing a disease state has beeninitiated or is likely to arise in the tested cells. In preferredembodiments, the methods include detecting the presence or absence ofthe relevant small molecule, in a sample of cells or extracellularmaterial from the subject, the presence or absence of a disease state.Preferably the sample of cells or extracellular material is obtainedfrom a body tissue suspected of comprising diseased cells. Thus, thepresent method provides information relevant to diagnosis of thepresence of a disease state. In one embodiment, the sample of cells iscomprised mainly of a particular cellular organelle, e.g., mitochondria,Golgi apparatus, nuclei, etc.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one reagent fordetecting a relevant small molecule, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a relevant smallmolecule.

C. Pharmacometabolomics

The invention also pertains to a method for predicting a subject'sresponse to a therapeutic agent. The method includes obtaining a smallmolecule profile from the subject, and comparing the small moleculeprofile of the subject to a known standard established for thetherapeutic agent as an indication of whether the subject would benefitfrom treatment with the therapeutic agent.

Agents, or modulators which alter levels of particular relevant smallmolecules, as identified by a screening assay described herein can beadministered to individuals to treat (prophylactically ortherapeutically) disorders associated with the relevant small molecules.In conjunction with such treatment, the pharmacometabolomics (i.e., thestudy of the relationship between an individual's metaboprint and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacometabolomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's metaboprint.Such pharmacometabolomics can further be used to determine appropriatedosages and therapeutic regimens. Accordingly, the small moleculeprofile of an individual can be determined to thereby select appropriateagent(s) for therapeutic or prophylactic treatment of the individual.

The known standard can be obtained from subjects who benefited from theagent, e.g., patients who were treated with the agent and were cured,maintained their health, or prevented or slowed the deterioration ofhealth. The known standard can be taken from a particular tissue, organ.It can also be taken from any organelle, cell, or cellular compartmentduring any point during the beneficial treatment. It can be derived froma single patient or from an average of more than one patient who weretreated successfully with the agent. In addition, the known standard canalso be derived using other techniques.

Pharmacometabolomics deals with clinically significant hereditary andnon-hereditary variations in the response to drugs due to altered drugdisposition and abnormal action in affected persons. In general, severaltypes of pharmacometabolomic conditions can be differentiated. Forexample, certain pharmacometabolomic conditions may be the result ofgenetic conditions. The genetic conditions may be transmitted as asingle factor altering the way drugs act on the body (altered drugaction) or genetic conditions transmitted as single factors altering theway the body acts on drugs (altered drug metabolism). Thesepharmacometabolomic conditions can occur either as rare defects or aspolymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency(G6PD) is a common inherited enzymopathy in which the main clinicalcomplication is haemolysis after ingestion of oxidant drugs(anti-malarials, sulfonamides, analgesics, nitrofurans) and consumptionof fava beans. Examples of non-hereditary conditions which may affectthe way drugs act on the body or the way the body acts on the drugsinclude the ingestion of certain drugs, the substance dependence of thepatient, the diet of the patient, non-hereditary medical conditions ofthe patient, etc.

The small molecule profile and metaboprint an individual can bedetermined to select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition,pharmacometabolomic studies can be used to identify an individual's drugresponsiveness metaboprint. This knowledge, when applied to dosing ordrug selection, can avoid adverse reactions or therapeutic failure andthus enhance therapeutic or prophylactic efficiency when treating asubject with an agent, such as an agent identified by one of theexemplary screening assays known in the art.

D. Monitoring of Effects During Clinical Trials

The invention also pertains to a method for metabolomically monitoringthe effectiveness of a therapeutic agent in clinical trials. The methodincludes obtaining a small molecule profile from a subject in a clinicaltrial being treated with a therapeutic agent, and monitoring changes inthe small molecule profile of the subject as an indication of theeffectiveness of the therapeutic agent in the subject. In oneembodiment, the small molecule profile of the subject can be compared toa predetermined standard.

Monitoring the influence of agents (e.g., drugs, therapeutic compounds)on the small molecule profile can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent determined by a screening assay as described herein toincrease levels of certain relevant small molecules, can be monitored inclinical trails of subjects exhibiting decreased levels of certain smallmolecules. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease levels of a certain relevant small molecule,can be monitored in clinical trails of subjects exhibiting increasedlevels of the certain relevant small molecule. In such clinical trials,the level of the certain small molecule and, preferably, the remainderof the small molecule profile can be used as a “read out” of the diseasestate of the particular cell.

For example, and not by way of limitation, small molecules that aremodulated in cells by treatment with an agent (e.g., compound, drug orsmall molecule) can be identified in screening assays. The effect ofagents on cellular proliferation disorders, for example, can be studiedin a clinical trial. For example, cells can be isolated and smallmolecule profiles of either whole cells or particular organelles can betaken. In this way, the small molecule profile can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

In an embodiment, the present invention provides a method for monitoringthe effectiveness of treatment of a subject with an agent (e.g.,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate identified by the screening assays described herein)comprising the steps of (i) obtaining a pre-administration sample from asubject prior to administration of the agent; (ii) detecting the smallmolecule profile of the preadministration sample; (iii) obtaining one ormore post-administration samples from the subject; (iv) detecting thesmall molecule profile of the post-administration samples; (v) comparingthe small molecule profile of the pre-administration sample with thesmall molecule profile of the post administration sample or samples; and(vi) altering the administration of the agent to the subjectaccordingly. For example, increased administration of the agent may bedesirable to increase the level of certain relevant small molecules tohigher levels than detected, i.e., to increase the effectiveness of theagent. Alternatively, decreased administration of the agent may bedesirable to decrease the level of certain relevant small molecules tolower levels than detected, i.e., to decrease the effectiveness of theagent.

12. Identification of Compounds Which Protect Mitochondrial Function

In a further embodiment, the samples are obtained from a specificorganelle, e g., mitochondria, nuclei, ribosomes, Golgi apparatus,endoplasmic reticulum, etc. The small molecule profiles obtained fromthe organelles can be used to identify small molecules which are ofparticular relevance to the particular organelle, in both health anddisease states. In one embodiment, the invention pertains to standardsmall molecule profiles of particular organelles, e.g., nuclei,mitochondria, chloroplasts, Golgi apparatus, endoplasmic reticulum,ribosomes, etc.

The term “standard small molecule profiles of particular organelles”includes the averaged profiles of particular organelles. The standardprofiles can be averaged over more than one profile from a particularindividual, or from a population. Furthermore, the profiles can all befrom the same type of cells (e.g., liver cells, muscle cells, nervecells, brain tissue cells, blood cells, immune system cells, etc.) ordifferent types of cells. The standard profiles can be taken of anyorganelle of interest. The organelle of interest can be obtained,generally, through methods known in the art, such as fractionalcentrifugation. Examples of organelles which can be analyzed throughthis method include, for example, nuclei, mitochondria, chloroplasts,centrioles, ribosomes, Golgi apparatus, endoplasmic reticulum, etc.

In one embodiment, the organelles are mitochondria. Mitochondrialdysfunction has been implicated in a wide range of physiologicalconditions, such as neurodegenerative diseases, aging processes,diabetes, and cancer. Mitochondria buffer intracellular calcium, areresponsible for production of ATP, and play a key role in cell deathpathways, such as apoptosis (Green and Reed, Science 281:1309-1312,1998; Susin et al., Biochim. Et. Biophys. Acta 1366:151-165, 1998). Dueto their central role in a wide array of cellular respiratory, oxidativeand metabolic processes, defects in mitochondrial activity impact therate of ATP production, calcium homeostasis, free radical production andrelease of apoptosis inducing factors. (Ernster and Schatz, J. CellBiol. 91:227s-255, 1981).

Therefore, in one embodiment, the invention pertains to a method foridentifying compounds relevant to mitochondrial related disorders. Inone embodiment, the invention pertains to small molecule profiles ofmitochondria which identify about 70% or more of the small molecules,about 75% or more of the small molecules, about 80% or more of the smallmolecules, about 85% or more of the small molecules, about 90% or moreof the small molecules, about 91% or more of the small molecules, about92% or more of the small molecules, about 93% or more of the smallmolecules, about 94% or more of the small molecules, about 95% or moreof the small molecules, about 96% or more of the small molecules, about98% or more of the small molecules, about 99% or more of the smallmolecules of the mitochondria. In another embodiment, the inventionpertains to methods for identifying compounds which are present inaberrant amounts when standard small molecule profiles of mitochondriaare compared to small molecule profiles of mitochondria afflicted with amitochondrial related disorder. The invention pertains to both thestandard mitochondria profile and the compounds identified to berelevant to mitochondria related disorders.

The term “mitochondrial related disorders” include disorders associatedwith processes associated with the mitochondria. Mitochondrial relateddisorders include neurodegenerative diseases, aging, diabetes, andcancer. Further more mitochondrial related diseases included thoserelated to the production of ATP, intracellular calcium, free radicals,and apopotosis.

In another embodiment, the compounds identified from the small moleculeprofile of the mitochondria are assayed for biologically active smallmolecules which protect mitochondrial function. Examples of assays thatcan be used for evaluating mitochondrial function include assays whichevaluate the inhibition of production of reactive oxygen species (e.g.,assays using dichlorofluorescin diacetate), assays for mitochondrialpermeability transition (MPT) (e.g., assays using dyes such as 2-4dimethylaminostyryl-N-Methlypyridinim); mitochodrial electrochemicalpotential gradients; and cell death assays with signals which can beused to induce insult, measure or release apoptogenic molecules,structural changes in cells, DNA changes, activation of caspases, andtranslocation of membrane components. Assays that evaluate effects ofcompounds on electron transport chain are also included (Parker et al.,Neurology 44: 1090-96, 1994; Miller et al., J. Neurochem. 67:1897,1996). In another embodiment, the compounds which are determined to bepresent in aberrant amounts in mitochondrial related disorders areassayed for protecting function. The invention also pertains to apharmaceutical composition comprising any compound identified by theassays described herein and a pharmaceutically acceptable carrier.

In a further embodiment, the biologically active small molecules arechemically modified to enhance their biological activity. It is known inthe art that through chemical modifications, one can enhance thebiological activity, stability, or otherwise modify a molecule to makeit more suitable as a therapeutic or nutriceutical agent.

13. Small Molecules Libraries and Methods of Use

In one embodiment, the invention pertains to the creation of smallmolecule libraries from cells, cellular compartments, and organelles,e.g., cells, cellular compartments, and organelles in health, diseased,and altered states. The small molecule libraries can be derived from thesame or different animal organs. For example, the small moleculelibraries can be derived from cells of the heart, brain, kidney, liver,done, blood, gastrointestinal tract, and/or muscle. In addition, thesmall molecule libraries can be derived from individuals suffering froma particular disease state, e.g., cardiovascular diseases,neurodegenerative diseases, diabetes, obesity, immunological disorders,etc.

The creation of the libraries involves fractionating cell components,preparing cellular extracts, and fractionating the small molecules bymethods such as HPLC, thin layer chromatography (TLC), electrochemicaltechniques and other methods known in the art for separating suchcompounds. The compounds can be also separated using their charge, mass,hydrophilicity, and hydrophobicity. Furthermore, the compounds can becharacterized using methods such as Mass Spectroscopy, NMR, IR, andother techniques known in the art for identifying organic compounds.

The term “library” includes searchable populations of small molecules ormixtures of molecules. In one embodiment, the library is comprised ofsamples or test fractions (either mixtures of small molecules orisolated small molecules) which are capable of being screened foractivity. For example, the samples could be added to wells in a mannersuitable for high throughput screening assays. In a further embodiment,the library could be screened for binding compounds by contacting thelibrary with a target of interest, e.g., a protein or a nucleic acid.

In further embodiment, the invention pertains to a library of compoundsfrom cells on a solid support, e.g., a solid support suitable forscreening assays, e.g., a solid support suitable for high throughputassays. The invention also pertains to a cellular small molecule librarypackaged in a container comprising the small molecule library, a solidsupport, and a label identifying the contents.

In yet another embodiment, the small molecule libraries are derived fromspecific cellular compartments, e.g., the cytoplasm, the nucleus, themitochondria, the choloroplast.

In one embodiment, the samples are screened for activity as a library.Within the last decade, small molecule libraries have been generatedusing combinatorial chemistry techniques to identify biologically activemolecules. For example, libraries of compounds have been screened forbiological activity using high throughput assays. For example,anti-tumor assays involve adding compounds to cancer cells in plasticwells and monitoring the effects of the compounds on cell survival. Thecompounds which effect cell survival, are identified as potential leadmolecules.

Libraries can be screened to determine whether any samples of thelibrary have a desired activity, and, if so, to identify the activecompound or sample. Methods of screening libraries have been described(see, e.g., Gordon et al., J Med. Chem.). Soluble small moleculelibraries can be screened by affinity chromatography with an appropriatereceptor to isolate ligands for the receptor, followed by identificationof the isolated ligands by conventional techniques (e.g., massspectrometry, NMR, and the like). Immobilized samples can be screened bycontacting the samples with a soluble receptor; preferably, the solublereceptor is conjugated to a label (e.g., fluorophores, calorimetricenzymes, radioisotopes, luminescent compounds, and the like) that can bedetected to indicate ligand binding. Alternatively, immobilized samplescan be selectively released and allowed to diffuse through a membrane tointeract with a receptor.

In vitro systems may be designed to identify the samples of thelibraries of the invention capable of interacting with targets ofinterest. The identified samples may contain useful compounds which may,for example, modulate the activity of the target; be useful inelaborating the biological function of the target; be utilized inscreens for identifying additional compounds that disrupt the normalinteractions of the target; or be useful themselves as disrupters ofsuch interactions.

The term “target” includes proteins and mixtures of proteins (e.g.,naturally occurring proteins, polypeptides, peptidomimitics, mutantproteins, and recombinant proteins). The term “target” also includesnucleic acid and mixtures of nucleic acids (e.g., RNA and DNA, bothnaturally occurring nucleic acids, synthesized nucleic acids, mutantnucleic acids, and recombinant nucleic acids) or lipids (e.g., membranesor membrane fragments). In one embodiment, the target is involved in adisease state of interest. Furthermore, it may be necessary to vary theconditions such that the target is able to maintain its cellularconfiguration.

The screening assays can be conducted in a variety of ways. For example,one method for identifying small molecules that interact with a targetor targets involves anchoring a target onto a solid phase, contacting itwith samples of small molecules, and detecting target/sample complexesanchored on the solid phase at the end of the reaction.

For example, microtiter plates may be used as the solid phase. Theanchored component may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished by simplycoating the solid surface with a solution of the sample or the targetand drying. The surfaces may be prepared in advance and stored. Covalentattachments include, for example, chemically linking the compound ortarget to the plate.

In one method of conducting the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed under conditionssuch that any target-sample complexes formed are capable of beingdetected. The complexes may be anchored on to the solid surface. Thedetection of complexes anchored on the solid surface can be accomplishedin a number of ways. Where the previously nonimmobilized component ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the previously nonimmobilizedcomponent is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the previously nonimmobilized component (the antibody, inturn, may be directly labeled or indirectly labeled with a labeledanti-Ig antibody).

In another embodiment, the invention provides a method (also referred toherein as a “screening assay”) for identifying targets which bind to thesamples of the library. It also includes methods for identifying samplesof the library which have a stimulatory or inhibitory effect on targets,for example, or target activity.

In an embodiment, the invention provides assays for screening librariesof the invention to identify samples which bind to or modulate theactivity of a target. Libraries of samples may be presented in solution(e.g., Houghten (1992) Biotechniques 13:412-421), on beads (Lam (1991)Nature 354:82-84), or chips (Fodor (1993) Nature 364:555-556).

For example, in one embodiment, the samples are prepared appropriatelyfor a interaction with a specific target using a high thoroughputscreen. The high throughput screen then is used to identify which of thesamples, bind or otherwise interact with the target.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a target (e.g., a protein of interest or a biologically activeportion thereof) is contacted with a sample of the library and theability of the sample to modulate the target's activity is determined.Determining the ability of the sample to modulate the target's activitycan be accomplished by methods suitable for the particular target.Determining the ability of the sample to modulate the ability of atarget to bind to its substrate can be accomplished, for example, bycoupling the substrate with a radioisotope or enzymatic label such thatbinding of the target to its substrate can be determined by detectingthe labeled substrate in a complex with the target. For example,substrates can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly orindirectly, and the radioisotope detected by direct counting ofradioemmission or by scintillation counting. Alternatively, substratescan be enzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a target substrate with the samples of theinvention and determining the ability of the samples to modulate (e.g.,stimulate or inhibit) the activity of the target.

Determining the ability of a target to bind to or interact with a targetsubstrate can be accomplished by one of the methods described above fordetermining direct binding. In an embodiment, determining the ability ofthe target to bind to or interact with its substrate can be accomplishedby determining the activity of the substrate. For example, the activityof the substrate can be determined by detecting induction of a cellularsecond messenger of the target, detecting catalytic/enzymatic activityof the target or its substrate, detecting the induction of a reportergene (comprising a target-responsive regulatory element operativelylinked to a nucleic acid encoding a detectable marker, e.g.,luciferase), or detecting a target-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which a target (e.g., a protein, a polypeptide, or anucleic acid) is contacted with a sample of the invention and theability of the samples to bind to the target is determined. Binding of asample to the target can be determined either directly or indirectly asdescribed above. In a further embodiment, the assay includes contactingthe target with a compound which is known to bind to the target to forman assay mixture, contacting the assay mixture with a sample of theinvention, and determining the ability of the sample to interact withthe target, wherein determining the ability of the sample to interactwith the target comprises determining the ability of the sample topreferentially bind to the target as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a targetis contacted with a library of compounds of the invention and theability of the compounds to modulate (e.g., stimulate or inhibit) theactivity of the target is determined. Determining the ability of thetest compound to modulate the activity of the target can beaccomplished, for example, by determining the ability of the target tobind to another molecule by one of the methods described above fordetermining direct binding. Determining the ability of the target tobind to another molecule can also be accomplished using a technologysuch as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S.and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al.(1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is atechnology for studying biospecific interactions in real time, withoutlabeling any of the interactants (e.g., BIAcore). Changes in the opticalphenomenon of surface plasmon resonance (SPR) can be used as anindication of real-time reactions between biological molecules.

In yet another embodiment, the cell-free assay involves contacting thetarget with a compound which is known to bind to the target to form anassay mixture, contacting the assay mixture with a sample of theinvention, and determining the ability of the sample to interact withthe target, wherein determining the ability of the sample to interactwith the target comprises determining the ability of the sample topreferentially bind to or modulate the activity of the target.

The libraries of compounds of the invention can also be screened usingcombinatorial methods such as those described in WO 99/31267 for thesimultaneous identification of novel biological targets and leadstructures for drug development.

14. Small Molecules Databases and Methods of Use

In one embodiment, the invention pertains to the creation of smallmolecule databases containing information regarding the metabolome ofcells, cellular compartments, and organelles, e.g., cells, cellularcompartments, and organelles in health, diseased, and altered states.The information regarding the small molecules of each cell, cellularcompartment, or organelle can be found using the separation andanalytical techniques described elsewhere in the application. The smallmolecule databases can include compounds derived from the same ordifferent animal organs. For example, the small molecule databases caninclude compounds obtained from cells of specific organs such as aheart, brain, kidney, liver, done, blood, gastrointestinal tract, and/ormuscle. In addition, the small molecule databases can includeinformation regarding compounds obtained from individuals suffering froma particular disease state, e.g., cardiovascular diseases,neurodegenerative diseases, diabetes, obesity, immunological disorders,etc.

The databases can be made based on information obtained from thetechniques described elsewhere in the application to determine theidentity and presence of various small molecules in cells, cellularcompartments, and organelles. The databases may include informationregarding the compounds found, such as structure, molecular weight,amounts found in particular organelles in a particular state of health,and any other information that a person of skill in the art wouldconsider relevant and useful to be contained in the database. Forexample, information regarding known biochemical pathways involving theparticular compound may also be included as well as other suchinformation.

In one embodiment, the databases of the invention contain information onthe compounds of the metabolome of a particular organelle of aparticular species in a particular state of health from a particularorgan (e.g., one database may include compounds of the metabolome of themitochondria of a healthy human heart). In other embodiments, thedatabases may include information regarding the metabolome of a varietyof organelles (e.g., mitochondria, nuclei, Golgi apparatus, endoplasmicreticulum, ribosomes, cytosol, chloroplasts, etc.) or cells from aparticular species from a particular organ in a particular state ofhealth. In another embodiment, the databases may include informationregarding either specific organelles or cells from a variety of tissues(e.g., fatty tissue, muscle tissue, nerve tissue, brain tissue, hearttissue, bone tissue, blood, connective tissue, retinal tissue, etc.)from an organism in a health or diseased stated (e.g, the tissue can befrom an organism suffering from any disorder known to afflict it).Examples of disorders include neurological disorders, central nervoussystem disorders, metabolic disorders, cardiovascular disorders,immunological disorders, oncological disorders. In a further embodiment,a database may comprise information regarding compounds of the entiremetabolome of a particular species, e.g., human, rat, mouse, dog, cat,etc.

If the database is in electronic form, the program used to organize thedatabase can be any program known in the art which is capable of storingthe information in a useful format.

The databases of the invention can be organized in such a way that theycan be licensed to companies, such as pharmaceutical companies. Thedatabases can then be used for many purposes, such as drug discovery,design, etc.

14. Methods of Identifying Biologically Active or Disease Relevant SmallMolecules from Cell Samples

In one embodiment, the invention pertains to yet another method foridentifying biologically active small molecules. This method includesobtaining cells from a tissue culture, an animal source or extracellularmaterial from a subject; obtaining samples of small molecules; testingthe samples for biological activity; and identifying samples which havebiological activity. The samples that are found to have biologicalactivity can then be further fractionated and profiled by methods knownin the art, such as HPLC, thin layer chromatography (TLC), andelectrochemical methods. The resulting compounds or fractions can thenbe retested for biological activity. If desired, the fractionation cancontinue until the individual compounds or mixtures of compounds withbiological activity are identified. These biologically active compoundscan then be used, for example, as lead compounds for drug design, usedas pharmaceutical or nutriceutical agents, labeled and used to identifyother components of pathways associated it, or used in otheradvantageous capacities.

The term “tissue culture or source” includes subjects, such as plants,bacteria, prokaryotes, eukaryotes, animals (e.g., yeast, mammals, e.g.,rats, mice, dogs, cats, primates (e.g., humans, chimpanzees, monkeys),horses, cattle, and bears). The subjects may be healthy, suffering froma disease state, or at risk of suffering from a disease state. Examplesof disease states include those which alter the amounts of various smallmolecules of the cell or cellular compartment (e.g., diabetes, cancer,AIDS, neurodegenerative disorders, etc.). The language “tissue cultureor source” includes cell lines such as can be found deposited with ATCC(e.g., cell lines corresponding to disease states, bacterial cell lines,animal cell lines, etc.). If necessary, the cells can be culturedaccording to methods and techniques known in the art (see, for example,Ausubel et al. Current Protocols in Molecular Biology (New York: JohnWiley & Sons). Other examples of sources include, for example, cellsfrom CAP23, Hela, human cell cultures, human placenta, lymphoblasts,mammalian muscle biopsies, rat brain, rat liver, and yeast.

In one embodiment, the samples are test fractions, fractionated bytechniques known in the art, such as, for example, HPLC (Kristal, et al.Anal. Biochem. 263:18-25 (1998)), thin layer chromatography (TLC), orother methods known in the art (Methods of Enzymology). The testfractions may be separated based on molecular weight. The test fractionscan then be screened for biological activity, before, after, or withoutfurther purification. The biologically active samples or test fractionscan then be further fractionated and characterized using methods suchas, for example, mass spectroscopy (Teusink, B. et al. MethodsMicrobiol., 26:297-336 (1998)), infra-red spectroscopy, and nuclearmagnetic resource (Brindle, K. et al. J. Mol. Recog. 10:182-187 (1997))to identify biologically active small molecules.

The term “biologically active small molecules” include small moleculeswhich modulate the activity of a biological system or pathway. Forexample, biologically active small molecules may be identified using invitro or in vivo assays known in the art. Biologically active smallmolecules can also be identified by screening assays against proteintargets which have been implicated in a disease state. In anotherembodiment, biologically active small molecules can be identified usingcell-based assays. For example, biologically active small molecules withanti-tumor activity can be identified, for example, by their effect onthe growth of a panel of tumor cell lines (Lillie et al. Cancer Res.53(13):3172-8 (1993)). Similarly, biologically active small moleculeswith neuronal protection activity may be identified by exposing primaryor cultured neurons to the compounds and toxic agents, such asglutamate, and identifying the compounds which protect the neurons fromdeath. Animal models can also be used to identify biologically activesmall molecules. For example, animal models of Huntington's Disease,Parkinson's disease, and ALS can be used to identify biologically activesmall molecules useful as neuroprotective agents. (Kilvenyi, Nature Med.5:347-350 (1999); Mathews et al, Experimental Neurology 157:142-149(1999)). In a further embodiment, the identified biologically activesmall molecules can then be chemically modified to further enhance theirpharmaceutical or nutriceutical properties.

The term “isolated” includes molecules (e.g., small molecules) which areseparated from other molecules which are present in the natural sourceof the molecules. In an embodiment, an “isolated” small molecule is freeof other molecules (both other small molecules and macromolecules) whichnaturally are present of the organism from which the small molecules arederived.

15. Use of Small Molecule Profiles for Tissue Typing and ForensicScience

The small molecule profiling of cells, cellular compartments, particularorganelles, and/or extracellular material of the present invention canalso be used to identify individuals, populations, or species fromminute biological samples. The method includes taking one or moresamples from a subject, population, or species and determining the smallmolecule profiles of the samples; taking a sample from a unknown sourceand determining its small molecule profile; and comparing the two smallmolecule profiles to determine whether the small molecule profiles arefrom the same individual, population or species.

It is expected that certain small molecules will be present in uniqueamounts in each person's cells, cellular compartments, organelles, orextracellular material. It is also expected that certain small moleculesmay be present in unique amounts in a particular population or species'cells, cellular compartments, organelles, or extracellular material. Byusing several of these compounds as markers, one could determine whetheror not a sample was or was not obtained from the same individual,population, or species as a reference sample. This method of tissuetyping can be used alone or in combination with more conventionaltechniques for determining the source of a tissue sample. Examples ofconventional techniques include RFLP (restriction fragment lengthpolymorphism, U.S. Pat. No. 5,272,057), DNA analysis (e.g., PCR), bloodtyping, etc.

In addition, a database can be created out of numerous individuals',populations', or species' small molecule profile, thus enabling thepositive identification of even a small tissue sample whose smallmolecule profile is registered with the database.

Small molecule profiling of cells, cellular organelles, andextracellular material can also be used in forensics. Forensic biologyis a scientific field employing, for example, genetic typing ofbiological evidence found at a crime scene as a means for positivelyidentifying a perpetrator of a crime. In traditional DNA-basedforensics, PCR technology is used to amplify DNA sequences taken fromvery small biological samples such as tissues, e.g., hair or skin, orbody fluids, e.g., blood, saliva, or semen found at a crime scene. Theamplified sequence can then be compared to a standard, thereby allowingidentification of the origin of the biological sample.

It is believed that the small molecule profile of each person's bodyfluids, cells, or cellular organelles, should contain unique amounts ofvarious small molecule.

Therefore, a small molecule profile of a given sample should yield aunique “fingerprint” of the perpetrator of the crime. Unlikeconventional techniques, the present invention allows for a quickprofile of the sample without the time consuming task of PCR. PCR isdependent on a multitude of repeated copies of the perpetrator's DNA,and therefore, its reliability is somewhat uncertain. The invention alsoencompasses methods for using the claimed small molecule profiles incombination with more conventional techniques, such as PCR, for enhancedsensitivity.

The small molecule profiles of the invention can also be used to developsmall molecule reagents to identify particular tissue types. Forexample, certain tissues (e.g., muscle, blood, urine, spinal fluid,interstial fluid, nervous tissue, fatty tissue, etc.) should have uniquesmall molecule profiles as compared to other body tissues. These tissueswill have enhanced concentrations of certain key small molecules andhave diminished concentrations of others. The identity of these tissuespecific small molecules may be consistent over a subset of thepopulation or the entire species as a whole.

Therefore, the invention pertains to the use of small molecule reagentsthat specifically react with key small molecules identified as beinglocalized to a specific tissue over a subset of the population. Thesmall molecule reagents can then be used to identify the identity of atissue of unknown origin (e.g., brain, blood, urine, spinal fluid,interstial fluid, muscle, fatty tissue, etc.).

16. Pharmaceutical Compositions

In another embodiment, the invention pertains to pharmaceuticalcompositions comprising a biologically active small molecule, diseaserelevant, or another molecule obtained through using the methods of theinvention and a pharmaceutically acceptable carrier. In anotherembodiment, the invention includes nutriceutical preparations ofbiologically active small molecules of the invention.

The biologically active small molecules may be chemically modified toenhance their biological activity. It is known in the art that throughchemical modifications, one can enhance the biological activity,stability, or otherwise modify a molecule to make it more suitable as apharmaceutical or nutriceutical agent.

The language “pharmaceutical composition” includes preparations suitablefor administration to mammals, e.g., humans. When the compounds of thepresent invention are administered as pharmaceuticals to mammals, e.g.,humans, they can be given per se or as a pharmaceutical compositioncontaining, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

The phrase “pharmaceutically acceptable carrier” is art recognized andincludes a pharmaceutically acceptable material, composition or vehicle,suitable for administering compounds of the present invention tomammals. The carriers include liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject agent from one organ, or portion of the body,to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the patient. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol; phosphate buffer solutions; and other non-toxiccompatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, α-tocopherol, and the like; and metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willgenerally be that amount of the compound which produces a therapeuticeffect. Generally, out of one hundred percent, this amount will rangefrom about 1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 per cent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol and glycerol monostearate; absorbents, such as kaolin andbentonite clay; lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and coloring agents. In the case of capsules, tabletsand pills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert dilutents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the activecompound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. If desired, the effective dailydose of the active compound may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical composition.

As set out above, certain embodiments of the present compounds cancontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” is art recognized and includes relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, e.g., Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci.66:1-19).

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesincludes relatively non-toxic, inorganic and organic base addition saltsof compounds of the present invention. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like.

The term “pharmaceutically acceptable esters” refers to the relativelynon-toxic, esterified products of the compounds of the presentinvention. These esters can be prepared in situ during the finalisolation and purification of the compounds, or by separately reactingthe purified compound in its free acid form or hydroxyl with a suitableesterifying agent. Carboxylic acids can be converted into esters viatreatment with an alcohol in the presence of a catalyst. Hydroxyls canbe converted into esters via treatment with an esterifying agent such asalkanoyl halides. The term also includes lower hydrocarbon groupscapable of being solvated under physiological conditions, e.g., alkylesters, methyl, ethyl and propyl esters. (See, for example, Berge etal., supra.)

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

17. Agricultural Methods of the Invention

In another embodiment of the invention, the invention includes a methodfor the identification of agents useful for agriculture, such as forexample, insecticides, pesticides, herbicides, and fertilizers.

Plants are an excellent source of small molecules. Many plant smallmolecules have been shown to have therapeutic benefit. Therefore, in oneembodiment, the invention pertains to a library of the small moleculesfrom plant extracts (e.g., extracts from a particular plant or part ofplant (e.g., seeds, flowers, berries, roots, sap, leaves, etc.), cellsfrom the plant, organelles (e.g., mitochondria, chloroplasts, nuclei,Golgi apparatus, etc.), cellular compartments, etc. These libraries canalso be screened for biologically active molecules using the methodsdescribed in previous sections. Furthermore, the plants also can beanalyzed using any of the separation or analytical techniques describedherein, e.g., HPLC, TLC, electrochemical analysis, mass spectroscopy,refractive index spectroscopy (RI), Ultra-Violet spectroscopy (UV),fluorescent analysis, radiochemical analysis, Near-InfraRed spectroscopy(Near-IR), Nuclear Magnetic Resonance spectroscopy (NMR), LightScattering analysis (LS) and other methods known in the art.

Furthermore, comparison of plant small molecule profiles could lead tothe identification of compounds which are relevant to the plant'sresistance of certain diseases or environmental conditions.

In addition, the method also pertains to small molecule profiles andsmall molecule libraries of plants. For example, the small moleculeprofiles can be used to determine plant deficiencies of certaincompounds, and analyze plant diseases in a method analogous to thecomparison of animal small molecule profiles. For example, a smallmolecule profile can be determined of a specific plant cell, cellcompartment or organelle (e.g., chloroplast, mitochondria, endoplasmicreticulum, Golgi apparatus, etc.). Standard plant cell profiles can alsobe generated. These can be compared to plants in particular diseasestates to determine which small molecules are present in aberrantamounts in the diseased cells.

In one method of the invention, small molecule profiles of insect cells,cellular compartments, or specific organelles are compared to smallmolecule profiles of insect cells, cellular compartments, or organellestreated with a known insecticide. The small molecule profiles can becompared to identify compounds which are relevant to the insecticideactivity. The compounds which are identified as relevant can then beidentified to further optimize the insecticidal activity of thecompounds.

The term “insecticides” include compounds which kill or other wise limitthe reproductive capacity of organisms from the order Isopoda (e.g.,Oniscus asellus, Armadillidium vulgare and Porcellio scaber, the orderDiplopoda (e.g., Blaniulus guttulatus), the order Chilopoda (e.g.,Geophilus carpophagus, Scutigera spec, etc.), the order Symphyla (e.g.,Scutigerella immaculata, etc.), the order Thysanura (e.g., Lepismasaccharina, the order Collembola (e.g., Onychiurus armatus), the orderOrthoptera (e.g., Blatta orientalis, Periplaneta americana, Leucophaeamaderae, Blattella germanica, Acheta domesticus, Gryllotalpa spp.,Locusta migratoria migratorioides, Melanoplus differentialis andSchistocerca gregaria, etc.), the order Dermaptera (e.g., Forficulaauricularia, etc.), the order Isoptera (e.g., Reticulitermes spp, etc.),the order Anoplura (e.g., Pediculus humanus corporis, Haematopinus spp.,Linognathus spp. etc.), the order Mallophaga (e.g., Trichodectes spp.Damalinea spp., etc.), the order Thysanoptera (e.g., Hercinothripsfemoralis, Thrips tabaci), the order Heteroptera (Eurygaster spp.,Dysdercus intermedius, Piesma quadrata, Cimex lectularius, Rhodniusprolixus and Triatoma spp., etc.), the order Homoptera (e.g., Aleurodesbrassicae, Bemisia tabaci, Trialeurodes vaporariorum, Aphis gossypii,Brevicoryne brassicae, Cryptomyzus ribis, Doralis fabae, Doralis pomi,Eriosoma lanigerum, Hyalopterus arundinis, Macrosiphum avenae, Myzusspp., Phorodon humuli, Rhopalosiphum padi, Phylloxera vastatrix,Pemphigus spp., Empoasca spp., Euscelis bilobatus, Nephotettixcincticeps, Lecanium corni, Saissetia oleae, Laodelphax striatellus,Nilaparvata lugens, Aonidiella aurantii, Aspidiotus hederae,Pseudococcus spp., Psylla spp., etc.), the order Lepidoptera, (e.g.,Pectinophora gossypiella, Bupalus piniarius, Cheimatobia brumata,Lithocolletis blancardelia, Hyponomeuta padella, Plutella maculipennis,Malacosoma neustria, Euproctis chrysorrhoea, Lymantria spp. Bucculatrixthurberiella, Phyllocnistis citrella, Agrotis spp., Euxoa spp., Feltiaspp., Earias insulana, Heliothis spp., Laphygma exigua, Mamestrabrassicae, Panolis flammea, Prodenia litura, Spodoptera spp.,Trichoplusia ni, Carpocapsa pomonella, Pieris spp., Chilo spp., Pyraustanubilalis, Ephestia kuehniella, Galleria mellonella, Tineolabisselliella, Tinea pellionella, Hofmannophila pseudospretella, Cacoeciapodana, Capua reticulana, Choristoneura fumiferana, Clysia ambiguella,Homona magnanima, Tortrix viridana, etc.), the order Coleoptera (e.g.,Anobium punctatum, Rhizopertha dominica, Bruchidius obtectus,Acanthoscelides obtectus, Hylotrupes bajulus, Agelastica alni,Leptinotarsa decemlineata, Phaedon cochleariae, Diabrotica spp.,Psylliodes chrysocephala, Epilachna varivestis, Atomaria spp.,Oryzaephilus surinamensis, Anthonomus spp., Sitophilus spp.,Otiorrhynchus sulcatus, Cosmopolites sordidus, Ceuthorrhynchusassimilis, Hypera postica, Dermestes spp., Trogoderma spp., Anthrenusspp., Attagenus spp., Lyctus spp., Meligethes aeneus, Ptinus spp.,Niptus hololeucus, Gibbium psylloides, Tribolium spp., Tenebrio molitor,Agriotes spp., Conoderus spp., Melolontha melolontha, Amphimallonsolstitialis Costelytra zealandica, etc.), the order Hymenoptera,(Diprion spp., Hoplocampa spp., Lasius spp., Monomorium pharaonis, Vespaspp., etc.), the order of the Diptera (e.g., Aedes spp., Anopheles spp.,Culex spp., Drosophila melanogaster, Musca spp., Fannia spp., Calliphoraerythrocephala, Lucilia spp., Chrysomyia spp., Cuterebra spp.,Gastrophilus spp., Hyppobosca spp., Stomoxys spp., Oestrus spp.,Hypoderma spp., Tabanus spp., Tannia spp., Bibio hortulanus, Oscinellafrit, Phorbia spp., Pegomyia hyoscyami, Ceratitis capitata, Dacus oleae,Tipula paludosa, etc.), the order Siphonaptera (e.g., Xenopsylla cheopisand Ceratophyllus spp., etc.), the order Arachnida (e.g., Scorpiomaurus, Latrodectus mactans, etc.), the order Acarina (e.g., Acarussiro, Argas spp., Ornithodoros spp., Dermanyssus gallinae, Eriophyesribis, Phyllocoptruta oleivora, Boophilus spp., Rhipicephalus spp.,Amblyomma spp., Hyalomma spp., Ixodes spp., Psoroptes spp., Chorioptesspp., Sarcoptes spp., Tarsonemus spp., Bryobia praetiosa, Panonychusspp., Tetranychus spp, etc.), Pratylenchus spp., Radopholus similis,Ditylenchus dipsaci, Tylenchulus semipenetrans, Heterodera spp.,Meloidogyne spp., Aphelenchoides spp., Longidorus spp., Xiphinema spp.,and Trichodorus spp.

In another embodiment, small molecule profiles of insect cells treatedwith a test compound can be compared to small molecule profiles ofinsect cells treated with a known insecticide to determine whether thetest compound may be an active insecticide.

The invention also pertains to insecticides comprising one or moreinsecticides identified by the methods of the invention. In oneembodiment, the insecticides of the invention are non-toxic to humans.

The insecticide compositions of the invention, both solids and liquids,may be applied to insect infestations or insect populations by spraying.The methods and equipment needed for a given treatment may be determinedby one skilled in the art. Furthermore, methods of the inventiondescribed herein may be used to treat insect infestations or populationsin dry, moist, or aquatic systems (e.g., the insect-infested area is aflowing or a standing body of water). An aquatic system which is treatedwith methods of the present invention may be either fresh water or saltwater. Furthermore, the insect control compositions of the invention maybe applied directly onto a host (e.g., an agricultural crop, aturfgrass).

EXEMPLIFICATION OF THE INVENTION Example 1

Method for Obtaining a Small Molecule Profile of a Cellular Compartment

Method for Obtaining a Small Molecule Profile of Mitochondria using HPLC

The following method demonstrates how small molecules are isolated frommitochondria for methods as described herein.

Mitochondrial Isolation

Mitochondria from a mammalian source are isolated by differentialcentrifugation in 140 mM KCI and 20 mM Hepes, pH 7.4. Following thefinal wash, mitochondria are resuspended in the same buffer and aliquotsare quick frozen in liquid nitrogen. Protein determinations are carriedout by Lowry using the Sigma Protein Assay Kit P5656).

Other mitochondrial samples are purified using a modified version of apublished protocol (Rigobello et al. (1995) Arch. Biochem. Biophys. 319,225-230). Mammalian liver mitochondria are obtained after decapitationof the subjects. The livers are dissected out and are placed in anice-cold solution containing 250 mM mannitol, 75 mM sucrose, 100 μMEDTA, 500 μM EGTA, and 10 mM Hepes (pH 7.4). The livers are homogenizedwith a motor-driven Teflon pestle and the homogenate is centrifuged at1000 g for 10 min. Supernatants are removed and centrifuged at 10,000 gfor 15 min. The pellets are washed in 250 mM mannitol, 75 mM sucrose,100 μM EDTA, 500 μM EGTA, and 10 mM Hepes (pH 7.4) with 0.5% bovineserum albumin (BSA) (Sigma A-6003). Following centrifugation, thepellets are then washed twice more in 250 mM mannitol, 75 mM sucrose, 30μM EDTA, and 10 mM Hepes (pH 7.4) with 0.5% BSA. Following the finalwash, mitochondria are resuspended in the 5 ml of the final bufferwithout BSA. An aliquot is removed, pelleted in a microfuge, washed oncewith 160 mM KCl, repelleted, and dry-frozen at −80° C. Samples areanalyzed by HPLC within 1 week of isolation.

HPLC Standards

Basic HPLC and Coulometric array methodology has been previouslydescribed with regard to their use for serum, urine, and tissue analysis(Beal et al. (1990) J. Neurochem. 55:1327; Matson et al. (1987) LifeSci. 41:905; LeWitt et al. (1992) Neurology 42:2111; Ogawa et al. (1992)Neurology 42:1702; Beal et al. (1992) J. Neurol. Sci. 108:80). Standardsfor stock solutions are obtained from Sigma and stored at −80° C. as 1mg/ml stocks in 20% MeOH containing 1% phosphoric acid. Subsequentdilution to working strength is made into 0.1 M NaCl. The assay sequenceis: standard, 8 samples, sample pool, standard, etc. Within-runprecision is derived from the prevision of the repeated pool assays.Precision varies primarily as a function of the level of the analyte andsecondarily as the complexity of the region in which it occurs.Typically at 5 pg precision is ±20%, at 500 pg ±5%, and at 1 ng ±3%.

Sample Preparation

Mitochondrial samples, including approximately 5 mg of mitochondrialprotein, are precipitated and extracted in 4 vol of acetonitrile, 4%acetic acid at −20° C. One milliliter of centrifuged supernatant isremoved, evaporated to dryness under vacuum, and reconstituted in 200 μlof mobile phase A (11 g/liter of pentane sulfonic acid at pH 3.00 withacetic acid). Recoveries, verified by sequential extractions andcomparisons of entire patterns, ranged from 93 to 100% for all compoundsresolved. This protocol conserves reactive species such as ascorbate andhomogentistic acid at 1 ng/ml concentrations. Reconstituted extractequivalent to 2 mg of mitochondrial protein is placed in an auto samplevial and immediately analyzed. Remaining extract is frozen at −80° C.for future confirmation analysis. Immediately prior to injection,samples are maintained in an autoinjector at 0-1° C.

Chromatographic Methods

To retain stability of retention times and response potentials, a mobilephase combination of mobile phase A (above) and mobile phase B (0.1 MLi-acetate at pH 3.00 with acetic acid in 80/10/10methanol/acetonitrile/isopropanol) is used. The chromatographic methodinvolves a 120-min complex gradient from 0% B to 100% B, with flow rateadjusted to compensate for aziotropic viscosity effects, and has beenpreviously described in detail (Milbury et al. (1997) in Progress inHPLC, Coulometic Electrode Array Detectors for HPLC, pp. 125-141, VSPInternational Science Publications). The mixed gradient is deliveredfrom a peak suppressor/gradient mixer to a PEEK-lined pulse damper priorto flowing through the auto sampler injector and on to two series C18columns [META250, 5-μm ODS, 250×4.6 mm I.D., ESA, Inc.].

The small molecules are detected using a 16-channel coulometricelectrode array (ESA, Inc., Model 5600 CEAS gradient system equippedwith a Kontron Model 460 autosampler) incremented from 0 to 900 mV in60-mV steps. Peak suppressor/gradient mixer, pulse damper, columns, anddetectors are contained within a temperature-controlled enclosuremaintained at 35° C. System functions are controlled by 5600-CEASsoftware installed on a 386 microcomputer.

The detected small molecules can then be analyzed on a computer tocreate a small molecule profile. The small molecule profile can then becompared, e.g., via subtraction, with small molecule profiles of othersamples. The isolated small molecules can then be used in assays knownin the art to determine biological activity.

Example 2

Method for Analyzing Metabolic Disorders Using Small Molecule Profiles

Method for Analyzing Differences in Small Molecule Profiles ofGenetically Altered Mice After Short Term Diet Variations

15 female C57B1/6J ob/ob mice and lean littermate controls (15 femaleC57B1/6J ?/+) and 15 male C57B1/Ks db/db mice and lean littermatecontrols (15 male C57B1/ks +/+) are obtained from Jackson labs at 4.5weeks of age, and are housed individually on normal mouse chow (West, D.B., 1992, Am. J. Physiol. 262:R1025-R1032) for 1 week prior to theinitiation of the study. The four groups of 15 mice each are thensacrificed by CO₂ euthanasia and tissues are then collected. Body weight(grams) of the four groups of mice at the time of sacrifice aremeasured. Small molecule profiles from cells from the hypothallumus arethen obtained from each group of mice and compared.

The mice (normal, lean, ob/ob, db/db, and/or tub/tub) are fed normallyprior to the initiation of the experiment, and then they are dividedinto one control and two experimental groups. The control group are thenmaintained on ad lib nourishment, while the first experimental group(“fasted group”) is fasted, and the second experimental group(“fasted-refed group”) is initially fasted, and then offered a highlypalatable meal shortly before the collection of tissue samples for smallmolecule profiling. Each test animal is weighed immediately prior to andimmediately after the experiment. Small molecule profiles are taken ofeach mouse from each group before and after the experiment. The profilesof each group are averaged and compared to those of different groups.

Method for Comparing Small Molecule Profiles of Mice After Long TermDiet Variations

Mice are fed normally prior to the initiation of the experiment, andthen are divided into one control and two experimental groups. Thecontrol group is then maintained on an ad lib diet of normal nourishmentin order to calculate daily food intake. The first experimental group(“underweight group”) is then underfed by receiving some fraction ofnormal food intake, 60-90% of normal, for example, so as to reduce andmaintain the group's body weight to some percentage, for example 80%, ofthe control group. The second experimental group (“overweight group”) isoverfed by receiving a diet which would bring the group to some levelabove that of the control, for example 125% of the control group. Tissuesamples are obtained for small molecule profiles to determine compoundswhich are present in different amounts in control versus overweightand/or underweight conditions.

Example 3

Comparison of Small Molecule Profiles of Different Types of Immune Cells

Method for Comparing the Small Molecule Profiles of TH1 and TH2 Cells

The transgenic T cell example is used to identify cellular smallmolecules present in TH2 cells. The identified small molecules are bepresent in different amounts in TH2 cells compared to TH1 cells.

Transgenic Mice

Naive CD4⁺ cells are obtained from the spleens and/or lymph nodes ofunprimed transgenic mouse strains harboring a T cell receptor (TCR)recognizing ovalbumin (Murphy et al., 1990, Science 250:1720).

Ova-Specific Transgenic T Cells

Suspensions of ova-specific T cells are co-cultured with stimulatorypeptide antigen and antigen presenting cells essentially as described inMurphy et al. (Murphy et al., 1990, Science 250:1720). Briefly, 2−4×10⁶T cells are incubated with approximately twice as many TA3 antigenpresenting cells in the presence of 0.3 μM Ova peptide. TH1 cultures maycontain approximately 10 ng/ml recombinant mIL-12. Conversely, TH2 cellsreceived IL-4 (1000 μ/ml). Cultures are harvested at various time pointsafter initiation of culture. T cells are purified of TA3 cells usinganti-CD4 coated magnetic beads (Dynal, Inc.). T cells are then pelletedby gentle centrifugation and lysed.

Tissue Collection:

Cells are then quick frozen on dry ice. Samples are homogenized togetherwith a mortar and pestle under liquid nitrogen.

Mitochondrial Isolation and Generation of Small Molecule Profiles

Cellular mitochondria are isolated and small molecule profiles aregenerated using the procedure given in Example 1.

Method for Comparing Small Molecule Profiles of Different TH CellSubpopulations

In this Example, the generation of small molecule profiles representingsmall molecules which are present in different amounts in TH cellsubpopulations and/or during the differentiation of such subpopulationsare described.

TH cell clones such as, D10.G4 (TH2), AE7 (TH1) and D1.1 (TH1), areused. Prior to stimulation, cell cultures are enriched for live cells bycentrifugation through a Ficoll gradient. Recovered cells are thencounted and their viability is examined using trypan blue exclusion.Cells are replated into either T25 or T75 flasks at approximately 5×10⁶cells in 5 mLs or 1.5×10⁶ cells in 10 mLs of culture medium,respectively.

Coating is then performed, generally, according to Current Protocols inImmunology, 1992, Coligan, J. E. et al., John Wiley & Sons, N.Y., pp3.12.4-3.12.6). Specifically, prior to plating, the flasks are coatedwith anti-CD3-ε antibodies (hybridoma supernatant from the 145-C11hybridoma; Parmingen, Inc., San Diego Calif.). For coating, antibodiesare resuspended in PBS at 1-2 μg/ml at a volume sufficient to coat thebottom of the flasks. Coating solution is incubated on the flasks for atleast one hour at 37° C.

After incubation, the antibody coating solution is removed by aspirationand cells will be immediately added. Flasks will then be placed in a 37°C. incubator for 6 hours. Cells are harvested by, for example, removalof supernatant from the culture. The mitochondria are removed from thecells by the procedure given above. Small molecule profiles of each typeof TH cell can then be done and analyzed to determine differences andsimilarities between the subpopulations.

Example 5

Method of Identifying Cardiovascular Disease Relevant Small Molecules

Method Using an Endolethial Cell Shear Stress Models to Obtain SmallMolecule Profiles and Identify Disease Relevant Small Molecules

Cell Culture

Primary cultures of HUVEC's are established from normal term umbilicalcords as described (In Progress in Hemostasis and Thrombosis, Vol. 3, P.Spaet, editor, Grune & Stratton Inc., New York, 1-28). Cells are grownin 20% fetal calf serum complete media (1989, J. Immunol. 142:2257-2263)and passaged 1-3 times before shear stress induction.

For induction, second passage HUVEC's are plated on tissueculture-treated polystyrene and subjected to 10 dyn/cm.sup.2 laminarflow for 1 and 6 hr. as described (1994, J. Clin. Invest. 94:885-891) or3-10 dyn/cm² turbulent flow as previously described (1986 Proc. Natl.Acad. Sci. U.S.A. 83:2114-2117).

Mitochondrial Isolation

Mitochondria from the HUVEC's are isolated by differentialcentrifugation in 140 mM KCI and 20 mM Hepes, pH 7.4. Following thefinal wash, mitochondria are resuspended in the same buffer and aliquotsare quick frozen in liquid nitrogen. Protein determinations are carriedout by Lowry using the Sigma Protein Assay Kit P5656).

Sample Preparation

Mitochondrial samples, including approximately 5 mg of mitochondrialprotein, are precipitated and extracted in 4 vol of acetonitrile, 4%acetic acid at −20° C. One milliliter of centrifuged supernatant isremoved, evaporated to dryness under vacuum, and reconstituted in 200 μlof mobile phase A (11 g/liter of pentane sulfonic acid at pH 3.00 withacetic acid). Reconstituted extract equivalent to 2 mg of mitochondrialprotein is placed in an auto sample vial and immediately analyzed.Immediately prior to injection, samples are maintained in anautoinjector at 0-1° C.

Chromatographic Methods

To retain stability of retention times and response potentials, a mobilephase combination of mobile phase A (above) and mobile phase B (0.1 MLi-acetate at pH 3.00 with acetic acid in 80/10/10methanol/acetonitrile/isopropanol) is used. The chromatographic methodinvolves a 120-min complex gradient from 0% B to 100% B, with flow rateadjusted to compensate for aziotropic viscosity effects, and has beenpreviously described in detail (Milbury et al. (1997) in Progress inHPLC, Coulometic Electrode Array Detectors for HPLC, pp. 125-141, VSPInternational Science Publications). The mixed gradient is deliveredfrom a peak suppressor/gradient mixer to a PEEK-lined pulse damper priorto flowing through the auto sampler injector and on to two series C18columns [META250, 5-μm ODS, 250×4.6 mm I.D., ESA, Inc.].

The small molecules are detected using a 16-channel coulometricelectrode array (ESA, Inc., Model 5600 CEAS gradient system equippedwith a Kontron Model 460 autosampler) incremented from 0 to 900 mV in60-mV steps. Peak suppressor/gradient mixer, pulse damper, columns, anddetectors are contained within a temperature-controlled enclosuremaintained at 35° C. System functions are controlled by 5600-CEASsoftware installed on a 386 microcomputer.

The detected small molecules can then be analyzed on a computer tocreate a small molecule profiles. The small molecule profiles of thecells are then compared to those not subjected to the turbulent flow.Small molecule present in aberrant amounts in the sample subjected tothe turbulent flow are identified for further investigation.

Example 6

Identification of Disease Relevant Small Molecules in Human Cell Samples

Human Tumor Example

In this example, a cell sample is taken from a malignant tumor in ahuman subject. Normal tissue is also collected from the subject from thesame or similar tissue as the tumor (e.g., normal breast tissue andbreast tumor tissue; normal prostate tissue and prostate tumor tissue,etc.). Normal tissue is also collected from a healthy subject from ananalogous tissue location.

The tissue samples are the homogenized and the mitochondria areisolated.

Mitochondrial Isolation

Mitochondria from a each tissue source are isolated by differentialcentrifugation in 140 mM KCI and 20 mM Hepes, pH 7.4. Following thefinal wash, mitochondria are resuspended in the same buffer and aliquotsare quick frozen in liquid nitrogen. Protein determinations are carriedout by Lowry using the Sigma Protein Assay Kit P5656).

Sample Preparation

The mitochondrial samples, including approximately 5 mg of mitochondrialprotein, are precipitated and extracted in 4 vol of acetonitrile, 4%acetic acid at −20° C. One milliliter of centrifuged supernatant isremoved, evaporated to dryness under vacuum, and reconstituted in 200 μlof mobile phase A (11 g/liter of pentane sulfonic acid at pH 3.00 withacetic acid). Reconstituted extract equivalent to 2 mg of mitochondrialprotein is placed in an auto sample vial and immediately analyzed.Immediately prior to injection, samples are maintained in anautoinjector at 0-1° C.

Chromatographic Methods

To retain stability of retention times and response potentials, a mobilephase combination of mobile phase A (above) and mobile phase B (0.1 MLi-acetate at pH 3.00 with acetic acid in 80/10/10methanol/acetonitrile/isopropanol) is used. The chromatographic methodinvolves a 120-min complex gradient from 0% B to 100% B, with flow rateadjusted to compensate for aziotropic viscosity effects, and has beenpreviously described in detail (Milbury et al. (1997) in Progress inHPLC, Coulometic Electrode Array Detectors for HPLC, pp. 125-141, VSPInternational Science Publications). The mixed gradient is deliveredfrom a peak suppressor/gradient mixer to a PEEK-lined pulse damper priorto flowing through the auto sampler injector and on to two series C18columns [META250, 5-μm ODS, 250×4.6 mm I.D., ESA, Inc.].

The small molecules are detected using a 16-channel coulometricelectrode array (ESA, Inc., Model 5600 CEAS gradient system equippedwith a Kontron Model 460 autosampler) incremented from 0 to 900 mV in60-mV steps. Peak suppressor/gradient mixer, pulse damper, columns, anddetectors are contained within a temperature-controlled enclosuremaintained at 35° C. System functions are controlled by 5600-CEASsoftware installed on a 386 microcomputer.

Analysis

The small molecule profiles of the healthy subject are compared to thesmall molecule profiles from the tumor tissue and the non-tumor tissueof the cancer patient. Small molecules which are present in aberrantamount in the tumor tissue are identified by comparing the profiles.

Example 7

Identification of ALS Relevant Small Molecules in Human Samples

The sample set consisted of about 50 blinded sera of an undisclosednumber of control and diagnosed amlotroptic lateral sclerosis (ALS)subjects. Within the control population of subjects, some subjects weresuffering from other central nervous system disease states. Thepopulation of ALS subjects included subjects who were newly discoveredto severely-impaired. In addition, the majority of ALS subjects weretaking Riluzole as their primary medication. All of the subjects were oncomplex drug regimens. All of the samples were examined for their totalchemical constituents, each constituent was scored for concentration andthe final results were databased.

At this point, half of the sera were unblinded as to their clinicalnature (control/non-control, age, gender, therapies, etc.). Using thishalf as reference, the database was examined for possible markers.General markers of the class of ALS subjects were identified as well assub-class markers within the population taking the drug Riluzole. FIG. 1shows markers which were identified in subjects with ALS who are nottaking Riluzole. FIG. 2 shows markers which were identified in subjectswith ALS who were taking Riluzole.

These markers were then used to predict the population affiliation ofthe remaining unblinded samples. The correct population affiliation wasdetermined for each of the samples using the markers. The metabolicsignificance of the markers we identified is currently underinvestigation. It is believed that the most prominent of these markersare not drug metabolites, but represent the successful metabolicresponse of the subject to the drug.

In this example, it was shown that the use of metabolomics can be usedto determine biochemical response of a subject to a drug as well as theunique biochemical characteristics of ALS subjects. FIG. 3 is a threedimensional graph showing the statistical separation of subjects. Thepopulation, ALS1, are suffering from ALS and are undergoing Riluzoletreatment, while the population ALS2 are suffering from ALS and notundergoing Riluzole treatment.

Example 8

Use of Databases Containing Metabolites and Other ALS Relevant SmallMolecules.

This example shows that databases can be created using metabolomics tosuccessfully determine whether or not a subject is suffering from aparticular disorder, such as, for example, ALS.

Databases of metabolites in the plasma of ALS patients and in controlswere generated. Metabolites in the plasma were separated using differentHPLC methods and detected by CEAS (coulometric electrode array system),LC/MS (liquid chromatograph/mass spectrometry) and/or GC/MS (gaschromatograph/mass spectrometry). Samples from controls and ALS patientswere profiled and data on each metabolite was extracted and stored inthe respective database.

Once the samples were collected, they were maintained in a frozen state.Each sample was thawed and immediately aliquoted into 100 μl portions,accessioned into LIMS and refrozen at −80. Subsequently, a known masswas homogenized in an equal volume of acidic acetone slush withPolyethylene Glycol 100 (PEG 100) and Dithiothreitol (DTT). Aliquotswere divided between each analysis platform and each preparation isdiluted (1:1) with a reference compound solution (RCS).

For LC sample preparation, the reference compound solution is composedof 20 reference compounds that are present at predefined concentrations.These reference compounds are all fluorescently-derivatized compounds ofvarying lipophilicity. The reference compounds are synthetically purecompounds, chosen for their chemical stability. In the case of GC samplepreparations, the RCS comprises straight chain hydrocarbons. Thechromatographic fluorescence profiles (LC) and paraffin profiles (GC)are used as the basis for a post-separation quality control (QC) on thechromatography. Furthermore, specific information from these profiles isused in the interpretation of the data files produced by otherinstrumentation.

A Surveyor HPLC fitted with a fluorescence detector and a bar-codereader is used. The effluent is split three ways with 2%, 2% and 96% ofthe stream being directed to two Thermo-Finnigan Mat-95 XP massspectrometers and an ESA 16 channel Coularray electrochemical detector,respectively. A single Thermo-Finnigan LTQ-FT mass spectrometer, whichhas an ion-trap (IT) front end and a Fourier-Transform (FT) backend, isset for monitoring both positive and negative ions respectively. Theelectrochemical detector allows one to see most electrochemically-activespecies with extreme sensitivity. Some compounds may be redundantlyvisualized across more than one of these machines. By using acombination of detectors, the vast majority of metabolites are detected.

The samples destined for GC are dried under vacuum desiccation for aminimum of 24 hours, before being derivatized under dried argon usingBistrimethyl-silyl-triflouroacetamide (BSTFA) catalyzed withderivatization reagent Trimethylsulfonyl chloride (TMSCl). A 5% phenylcolumn with a temperature range from 40° to 300° C. is used. Samples areanalyzed on a Thermo-Finnigan Mat-95 XP using Electron Impactionization, and high resolution. The resulting spectra are used foranalysis of elemental composition and identification.

Samples for induced coupling plasma/mass spectroscopy (ICP/MS) are aciddigested for 24 hours, filtered and separated by ion chromatographyprior to the introduction of the column effluent into the plasma of theICP/MS.

The methods described in this example identify, quantify, and store forstatistical analysis, peaks representative of unknown compounds inaddition to the known compounds/peaks. Where a peak is identified asstatistically significant, i.e. a biomarker to a definable population orsub-population, it is subjected to a chemical identification processusing many of the instruments used in the original analysis. Forexample, if the unknown peak is seen in the LC, the ion-trap (IT)portion is used to do a detailed fragmentation analysis of the molecule.If needed, repeated fragmentation cycles in the IT will be conducted. Ifthe unknown peak is seen in the GC, additional fragment analyses aredone at a higher resolution than normal (profiling resolution ˜20,000,compound identification resolution ˜100,000).

Several mathematical tools were employed to differentiate between thedatabases of data generated for the ALS and control groups. Mathematicaltools used for data analysis included partial least squares-discriminantanalysis (PLS-DA), “relative class association/weighted voting”, andscatter analysis (Multivariate Statistical Methods: A Primer: BryanManley, CRC Press; Kennedy R et al. Solving data Mining Problems throughPattern Recognition, Prentice Hall PTR; Erikson I et al. Multi andMegavariate Analysis: Principles and Applications. Umetrics, Umea,Sweden, 2001 edition). These analytical approaches were used to separateALS patients from controls and derive an initial metabolic signature forALS based on significant differences in their metabolomes. Thesesignatures highlight metabolites that are found at significantly higheror lower concentrations in ALS, marking the disease.

Partial least square discriminate analysis (PLS-DA) is similar to themore widely known principal components analysis. However, rather thanfinding independent components that best explain overall variance in adata set, PLS-DA finds components that best explain the differencesbetween two classes. These components can then be used to predict theclass membership of new examples.

Relative class association/weighted voting was first used to distinguishtwo types of leukemia based on gene expression profiles (Golub T, et al.Science 1999;286:531). This method begins by computing a “relative classassociation” for each compound, which is a measure of the degree towhich the concentration of the compound is associated with one of twoclasses of interest. The method then determines by a permutation testwhich compounds have significant relative class associations, and usesthese compounds to “vote” for membership in one of the two classes, andmay also use cross-validation on the training set to further prune thecompounds considered. The vote of each compound is weighted by itsrelative class association. The sample is assigned to one of the classesaccording to the sum of the votes.

Scatter analysis first detects compounds that have means that aresignificantly higher or lower among a target class (for example ALS)than among controls. The cutoff is usually 5 standard deviations in thecontrol distribution above or below the mean concentration for controls.For prediction, the presence of one of these compounds with aconcentration that is highly different from the mean in controls istaken as evidence that the source sample is not in the control class.

FIG. 4 shows that using PLS-DA, ALS patients were distinguishable fromtheir control counterparts. Fifteen patients with SALS had uniquefeatures that separated them from the eight control counterparts.

The databases generated can also be used to distinguish betweencontrols, subjects with SALS, and subjects with pure lower motor neurondisease. In this example, a set of 59 blinded plasma samples containedan undisclosed number of controls, subjects diagnosed with SALS, and afew patients with pure lower motor neuron disease. Each chemicalconstituent was scored for concentration and the final results werestored in a database. There were no significant differences in weight,age, or gender distribution between the ALS, and the control cohort,although more subjects with ALS took antioxidants than the controlcohort (85% vs. 30%, p<0.00001). After blinded data analysis,information on chromatographic peaks was extracted from the rawchromatographic data files and placed in a database. The clinical originof 35 of the 59 samples was unblinded and scatter analysis was used todetermine which compounds were at higher concentrations in ALS patientsthan in the controls.

The three statistical analysis approaches were then employed to evaluatedifferences in metabolite levels in ALS versus controls, and a set ofclass predictors was constructed from the unblinded set of samples.Compounds which are significantly different between ALS and controlswere discovered by algorithms and used to build “class predictors”. Theclass predictors used the concentrations of these informative compoundsto distinguish the profiles of ALS patients from profiles of individualsin the control groups. Broadly speaking, this is “supervised learning”,a kind of data mining in which a learning algorithm is presented withexamples of two or more “classes” and then attempts to derive a set ofrules (a class predictor) that will predict the class membership of newexamples. Thus, the supervised learning algorithm searches the profilesto find the compounds that best let it distinguish ALS profiles fromnon-ALS profiles, and base its class predictor on these compounds.

Subsequently, the disease status of the rest of the 24 blinded sampleswas predicted using the methods of the invention. Assignment of 11/12 ofthe control samples was done correctly, 8/8 of the ALS patients onRiluzole were correctly assigned and 3/4 of the ALS patients not takingRiluzole were also correctly assigned. An illustration of chromatogramsgenerated using the HPLC-CEAS is shown in FIG. 5.

After complete unblinding, a full analysis of the data set was done andthe results are shown in FIGS. 6A-6C. Using the peaks identified asconsistently variant (from controls) in one or more populations (FIGS.6A and 6B), metabolometric analysis was used to determine which subjectswere suffering from ALS and which subjects were on Riluzole therapy. Inaddition, subjects with pure lower motor neuron disease and SALS weredifferentiated. After unblinding, it was possible to identify fourgroups: controls (Black), ALS subjects on Riluzole (Blue), ALS patientsoff Riluzole (Red), and atypical ALS (Yellow) (FIG. 6C).

Three out of the four samples represented under atypical ALS turned outto be patients with pure lower motor neuron disease. The fourth had along disease course (>9 years). A series of endogenous cellularmetabolites induced by the drug Riluzole were identified. Thesecompounds may be a response to the drug and may be a part of themechanism of action of Riluzole or part of its side effects.

In this example it was shown that metabolomics can be used to accuratelydistinguish between subjects suffering from ALS and control subjects.The example shows that unique chemical markers can be discovered bystudying the databases using analytical techniques. This example alsoshows that the chemical markers discovered using the analyticaltechniques can successfully be used to diagnosis and distinguish betweengroups of subjects suffering from or not suffering from a particulardisorder, such as ALS.

INCORPORATION BY REFERENCE

The entire contents of all references and patents cited herein arehereby incorporated by reference. The entire contents of U.S. Pat. No.5,908,609 and all its references also expressly incorporated herein.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

The invention claimed is:
 1. A method for metabolomically identifyingsmall molecules indicative of amyotrophic lateral sclerosis, comprising:obtaining a small molecule profile from a subject suffering fromamyotrophic lateral sclerosis; and comparing the small molecule profilefrom the subject to a standard small molecule profile, therebyidentifying small molecules indicative of amyotrophic lateral sclerosis,wherein said small molecule profile is obtained using one or moretechniques which detect 50% or more of the small molecules in saidsample.
 2. The method of claim 1, wherein said subject is a human. 3.The method of claim 1, wherein said small molecule profile is obtainedfrom said subject's tissue.
 4. The method of claim 1, wherein said smallmolecule profile is obtained using one or more of the following: HPLC,TLC, electrochemical analysis, mass spectroscopy, refractive indexspectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent analysis,gas chromatography (GC), radiochemical analysis, Near-InfraRedspectroscopy (Near-IR), Nuclear Magnetic Resonance spectroscopy (NMR),and Light Scattering analysis (LS).
 5. A method for metabolomicallyidentifying small molecules indicative of Parkinson's disease,comprising: obtaining a small molecule profile from a subject sufferingfrom Parkinson's disease; and comparing the small molecule profile fromthe subject to a standard small molecule profile, thereby identifyingsmall molecules indicative of Parkinson's disease, wherein said smallmolecule profile is obtained using one or more techniques which detect50% or more of the small molecules in said sample.
 6. A method formetabolomically identifying small molecules indicative of schizophrenia,comprising: obtaining a small molecule profile from a subject sufferingfrom schizophrenia; and comparing the small molecule profile from thesubject to a standard small molecule profile, thereby identifying smallmolecules indicative of schizophrenia, wherein said small moleculeprofile is obtained using one or more techniques which detect 50% ormore of the small molecules in said sample.
 7. A method formetabolomically identifying small molecules indicative of Parkinson'sdisease, comprising: obtaining a small molecule profile from a subjectsuffering from Parkinson's disease; and comparing the small moleculeprofile from the subject to a standard small molecule profile, therebyidentifying small molecules indicative of Parkinson's disease, whereinsmall molecule profile is obtained using one or more techniques whichdetect 50% or more of the small molecules in said sample.
 8. A methodfor metabolomically identifying small molecules indicative ofdepression, comprising: obtaining a small molecule profile from asubject suffering from depression; and comparing the small moleculeprofile from the subject to a standard small molecule profile, therebyidentifying small molecules indicative of depression, wherein said smallmolecule profile is obtained using one or more techniques which detect50% or more of the small molecules in said sample.
 9. A method formetabolomically identifying small molecules indicative of schizophrenia,comprising: obtaining a small molecule profile from a subject sufferingfrom schizophrenia; and comparing the small molecule profile from thesubject to a standard small molecule profile, thereby identifying smallmolecules indicative of schizophrenia, wherein small molecule profile isobtained using one or more techniques which detect 50% or more of thesmall molecules in said sample.
 10. The method of claim 4, wherein saidsmall molecule profiles is obtained from said subject's cells, cellularorganelles, interstitial fluid, or saliva.
 11. The method of claim 5,wherein said subject is a human.
 12. The method of claim 5, wherein saidsmall molecule profiles is obtained from said subject's tissue.
 13. Themethod of claim 5, wherein said small molecule profiles is obtained fromsaid subject's cells, cellular organelles, interstitial fluid, orsaliva.
 14. The method of claim 5, wherein said small molecule profilesis obtained using one or more of the following: HPLC, TLC,electrochemical analysis, mass spectroscopy, refractive indexspectroscopy (RI), Ultra-Violet spectroscopy (UV), fluorescent analysis,radiochemical analysis, Near-InfraRed spectroscopy (Near-IR), NuclearMagnetic Resonance spectroscopy (NMR), gas chromatography (GC) and LightScattering analysis (LS).
 15. The method of claim 6, wherein saidsubject is a human.
 16. The method of claim 6, wherein said smallmolecule profiles is obtained from said subject's tissue.
 17. The methodof claim 6, wherein said small molecule profiles is obtained from saidsubject's cells, cellular organelles, interstitial fluid, or saliva. 18.The method of claim 6, wherein said small molecule profiles is obtainedusing one or more of the following: HPLC, TLC, electrochemical analysis,mass spectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), gas chromatography (GC) and Light Scatteringanalysis (LS).
 19. The method of claim 7, wherein said subject is ahuman.
 20. The method of claim 7, wherein said small molecule profilesis obtained from said subject's tissue.
 21. The method of claim 7,wherein said small molecule profiles is obtained from said subject'scells, cellular organelles, interstitial fluid, or saliva.
 22. Themethod of claim 7, wherein said small molecule profiles is obtainedusing one or more of the following: HPLC, TLC, electrochemical analysis,mass spectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), gas chromatography (GC) and Light Scatteringanalysis (LS).
 23. The method of claim 8, wherein said subject is ahuman.
 24. The method of claim 8, wherein said small molecule profilesis obtained from said subject's tissue.
 25. The method of claim 8,wherein said small molecule profiles is obtained from said subject'scells, cellular organelles, interstitial fluid, or saliva.
 26. Themethod of claim 8, wherein said small molecule profiless is obtainedusing one or more of the following: HPLC, TLC, electrochemical analysis,mass spectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), gas chromatography (GC) and Light Scatteringanalysis (LS).
 27. The method of claim 9, wherein said subject is ahuman.
 28. The method of claim 9, wherein said small molecule profilesis obtained from said subject's tissue.
 29. The method of claim 9,wherein said small molecule profiles is obtained from said subject'scells, cellular organelles, interstitial fluid, or saliva.
 30. Themethod of claim 9, wherein said small molecule profiles is obtainedusing one or more of the following: HPLC, TLC, electrochemical analysis,mass spectroscopy, refractive index spectroscopy (RI), Ultra-Violetspectroscopy (UV), fluorescent analysis, radiochemical analysis,Near-InfraRed spectroscopy (Near-IR), Nuclear Magnetic Resonancespectroscopy (NMR), gas chromatography (CC) and Light Scatteringanalysis (LS).
 31. The method of claim 4, wherein said small moleculeprofile is obtained from said subject's biological fluids.
 32. Themethod of claim 1, wherein said small molecule profile is obtained usingmass spectroscopy.
 33. The method of claim 1, wherein said smallmolecule profile is obtained using gas chromatography (GC).
 34. Themethod of claim 1, wherein said small molecule profile is obtained usingNuclear Magnetic Resonance spectroscopy (NMR).
 35. The method of claim1, wherein said small molecule profile is obtained using HPLC.
 36. Themethod of claim 4, wherein said small molecule profile is obtained fromsaid subject's spinal fluid.
 37. The method of claim 4, wherein saidsmall molecule profiles are obtained from said subject's serum.
 38. Themethod of claim 4, wherein said small molecule profile is obtained fromsaid subject's urine.
 39. The method of claim 5, wherein said smallmolecule profile is obtained from said subject's biological fluids. 40.The method of claim 14, wherein said small molecule profile is obtainedusing mass spectroscopy.
 41. The method of claim 14, wherein said smallmolecule profile is obtained using gas chromatography (GC).
 42. Themethod of claim 14, wherein said small molecule profile is obtainedusing Nuclear Magnetic Resonance spectroscopy (NMR).
 43. The method ofclaim 14, wherein said small molecule profile is obtained using HPLC.44. The method of claim 5, wherein said small molecule profiles areobtained from said subject's serum.
 45. The method of claim 5, whereinsaid small molecule profile is obtained from said subject's urine. 46.The method of claim 5, wherein said small molecule profile is obtainedfrom said subject's spinal fluid.
 47. The method of claim 6, whereinsaid small molecule profile is obtained from said subject's biologicalfluids.
 48. The method of claim 18, wherein said small molecule profileis obtained using mass spectroscopy.
 49. The method of claim 18, whereinsaid small molecule profile is obtained using gas chromatography (GC).50. The method of claim 18, wherein said small molecule profile isobtained using Nuclear Magnetic Resonance spectroscopy (NMR).
 51. Themethod of claim 18, wherein said small molecule profile is obtainedusing HPLC.
 52. The method of claim 6, wherein said small moleculeprofile is obtained from said subject's spinal fluid.
 53. The method ofclaim 6, wherein said small molecule profiles are obtained from saidsubject's serum.
 54. The method of claim 6, wherein said small moleculeprofile is obtained from said subject's urine.
 55. The method of claim7, wherein said small molecule profile is obtained from said subject'sbiological fluids.
 56. The method of claim 22, wherein said smallmolecule profile is obtained using mass spectroscopy.
 57. The method ofclaim 22, wherein said small molecule profile is obtained using gaschromatography (GC).
 58. The method of claim 22, wherein said smallmolecule profile is obtained using Nuclear Magnetic Resonancespectroscopy (NMR).
 59. The method of claim 22, wherein said smallmolecule profile is obtained using HPLC.
 60. The method of claim 7,wherein said small molecule profile is obtained from said subject'sspinal fluid.
 61. The method of claim 7, wherein said small moleculeprofiles are obtained from said subject's serum.
 62. The method of claim7, wherein said small molecule profile is obtained from said subject'surine.
 63. The method of claim 8, wherein said small molecule profile isobtained from said subject's biological fluids.
 64. The method of claim26, wherein said small molecule profile is obtained using massspectroscopy.
 65. The method of claim 26, wherein said small moleculeprofile is obtained using gas chromatography (GC).
 66. The method ofclaim 26, wherein said small molecule profile is obtained using NuclearMagnetic Resonance spectroscopy (NMR).
 67. The method of claim 26,wherein said small molecule profile is obtained using HPLC.
 68. Themethod of claim 8, wherein said small molecule profile is obtained fromsaid subject's spinal fluid.
 69. The method of claim 8, wherein saidsmall molecule profiles are obtained from said subject's serum.
 70. Themethod of claim 8, wherein said small molecule profile is obtained fromsaid subject's urine.
 71. The method of claim 9, wherein said smallmolecule profile is obtained from said subject's biological fluids. 72.The method of claim 30, wherein said small molecule profile is obtainedusing mass spectroscopy.
 73. The method of claim 30, wherein said smallmolecule profile is obtained using gas chromatography (GC).
 74. Themethod of claim 30, wherein said small molecule profile is obtainedusing Nuclear Magnetic Resonance spectroscopy (NMR).
 75. The method ofclaim 30, wherein said small molecule profile is obtained using 1{PLC.76. The method of claim 9, wherein said small molecule profile isobtained from said subject's spinal fluid.
 77. The method of claim 9,wherein said small molecule profiles are obtained from said subject'sserum.
 78. The method of claim 9, wherein said small molecule profile isobtained from said subject's urine.